3-Nitrogen-6, 7-dioxygen steroids and uses related thereto

ABSTRACT

A compound of the formula  
                 
 
and pharmaceutically acceptable salts, solvates, stereoisomers and prodrugs thereof, in isolation or in mixture, wherein, independently at each occurrence: R 1  and R 2  are selected from hydrogen, oxygen so as to form nitro or oxime, amino, sulfate, and sulfonic acid, and organic groups having 1-30 carbons and optionally containing 1-6 heteroatoms selected from nitrogen, oxygen, phosphorous, silicon, and sulfur, where R 1  and R 2  may, together with the N to which they are both bonded, form a heterocyclic structure that may be part of an organic group having 1-30 carbons and optionally containing 1-6 heteroatoms selected from nitrogen, oxygen and silicon, and where R 1  may be a 2, or 3 atom chain to numeral 2 so that —N—R 1 — forms part of a fused bicyclic structure to ring A; R 3  and R 4  are selected from direct bonds to 6 and 7 respectively so as to form carbonyl groups, hydrogen, or a protecting group such that R 3  and/or R 4  is part of hydroxyl or carbonyl protecting group; numerals 1 through 17 each represent a carbon having substitution as described. The compounds may be formulated into pharmaceutical compositions, and used in the treatment and/or prevention of various conditions, including inflammation, asthma, an allergic disease, chronic obstructive pulmonary disease, atopic dermatitis, solid tumors, AIDS, ischemia, and cardiac arrhythmias.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/845,775, filed Apr. 30, 2001; which application claims the benefit ofU.S. Provisional Patent Application No. 60/200,617 filed Mar. 28, 2000,where these applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

This invention is directed towards 3-nitrogen-6,7-dioxygenated steroids,compositions including these steroids, and therapeutic uses relatedthereto.

BACKGROUND OF THE INVENTION

The Inflammatory Response (Inflammation)

Inflammation is an essential localized host response to invadingmicroorganisms or tissue injury which involves cells of the immunesystem. The classic signs of inflammation include redness (erythema),swelling (edema), pain and increased heat production (pyrema) at thesite of injury. The inflammatory response allows the body tospecifically recognize and eliminate an invading organism and/or repairtissue injury. Many of the acute changes at the site of inflammation areeither directly or indirectly attributable to the massive influx ofleukocytes (e.g., neutrophils, eosinophils, lymphocytes, monocytes)which is intrinsic to this response. Leukocytic infiltration andaccumulation in tissue results in their activation and subsequentrelease of inflammatory mediators such as LTB₄, prostaglandins, TNF-α,IL-1β, IL-8, IL-5, IL-4, histamine, proteases and reactive oxygenspecies for example.

Normal inflammation is a highly regulated process that is tightlycontrolled at several levels for each of the cell types involved in theresponse. For example, expression of the pro-inflammatory cytokine TNF-αis controlled at the level of gene expression, translation,post-translational modification and release of the mature form from thecell membrane. Many of the proteins up-regulated during inflammation arecontrolled by the transcription factor, NF-κB. Pro-inflammatoryresponses are countered in some instances by endogenousanti-inflammatory mechanisms such as generation of IL-10. Acharacteristic of a normal inflammatory response is that it is temporaryin nature and is followed by a resolution phase which brings the stateof the tissue back to its prior condition. The resolution phase isthought to involve up-regulation of anti-inflammatory mechanisms, suchas IL-10, as well as down-regulation of the pro-inflammatory processes.

Inflammatory Disease

Inflammatory disease occurs when an inflammatory response is initiatedthat is inappropriate and/or does not resolve in the normal manner butrather persists and results in a chronic inflammatory state.Inflammatory disease may be systemic (e.g., lupus) or localized toparticular tissues or organs and exerts an enormous personal andeconomic burden on society. Examples of some of the most common andproblematic inflammatory diseases include asthma, allergy, rheumatoidarthritis, inflammatory bowel disease, psoriasis, emphysema, colitis,graft vs host disease, contact dermatitis, and ischemia-reperfusioninjury. Other disease states such as immunodeficiency diseases are nowknown to be associated with altered regulation of the chemokine/cytokinenetwork and their receptors, which can alter viral replication and AIDSpathogenesis.

Many of the tissue, cellular and biochemical processes which areperturbed in inflammatory disease have been elucidated and this hasallowed the development of experimental models or assays to mimic thedisease state. These in-vitro and in-vivo assays enable selection andscreening of compounds with a high probability of therapeutic efficacyin the relevant inflammatory disease. For example, the ability of acompound to inhibit the allergen-induced accumulation of inflammatorycells such as eosinophils and lymphocytes in the lavage fluid obtainedfrom sensitized animals is indicative of anti-asthma activity. Inparticular, this model system is useful in the evaluation of the effectsof compounds in the treatment of the late phase response andhyper-responsiveness that is characteristic of asthma, when lunginflammation is apparent.

Asthma and Allergy

Asthma and allergy are closely related with good evidence from clinicalstudies demonstrating a strong correlation between the severity ofasthma and the degree of atopy (allergy). Sensitization to allergens isbelieved to be the most important risk factor for asthma in bothchildren and adults, with approximately 90% of asthma cases exhibitingatopy.

Allergy is characterized by an increased blood serum IgE (antibody)level. Repeated exposure to allergens, in a process calledsensitization, is normally required to trigger atopy and the subsequentasthmatic or allergic response. Once B cells are exposed to allergens,they produce antibodies which bind to the surface of mast cells. Thecrosslinking of two antibodies by the antigen causes a series ofreactions resulting in degranulation and the release of a number ofmediators which modulate the inflammatory response. Mediators that arereleased or generated during the asthmatic and allergic response includehistamine, leukotrienes, prostaglandins, cytokines and tryptase.

Asthma is characterized by hyperresponsiveness of the airways, episodicperiods of bronchospasm and chronic inflammation of the lungs.Obstruction of the airways is reversible with time or in response todrug therapies. Patients exhibiting normal airflow may be hyperreactiveto a variety of naturally occurring stimuli, e.g., cold air, exercise,chemicals and allergen. The most common event initiating an asthmaticresponse is an immediate hypersensitivity to common allergens includingragweed pollen, grass pollen, various fungi, dust mites, cockroaches anddomestic animals. The symptoms of the disease include chest tightness,wheezing, shortness of breath and coughing. Asthma incidence andmortality has been increasing worldwide, doubling over the past 20 yearsdespite modern therapies.

The responses of the airways to allergen is complex and consists of anearly asthmatic response (EAR) which peaks 20-30 min after exposure tothe stimuli, is characterized by bronchoconstriction and normallyresolves after 1½ to 2 hours. The late asthmatic response (LAR)generally occurs 3-8 hours after initial exposure, and involves bothbronchoconstriction and the development of inflammation and edema in thelung tissue. This inflammation often becomes chronic, with epithelialdamage occurring and infiltration of the lungs with inflammatory cellssuch as eosinophils and neutrophils.

Current Treatments for Asthma

Glucocorticoids (steroids) are the most effective long-term therapy forthe treatment of asthma. For example, due to the presence of airwayinflammation even in mild asthma, inhaled steroids are used even inearly stage drug therapy. Although steroids are effectiveanti-inflammatories they are not very useful for the control of acuteasthma attacks. Orally delivered steroids are associated withsignificant side-effects and consequently their chronic use in thecontrol of asthma is minimal. Combination therapy is often employed fororally delivered steroids, where combination therapy may be divided intothe following areas: anti-inflammatory drugs (e.g., inhaled and oralsteroids), bronchodilators, (e.g., β₂-agonists, xanthines,anticholinergics), and mediator inhibitors (e.g., cromolyns andleukotriene antagonists). In general, moderate to severe asthma patientsare poorly served by the present armamentarium of drugs. Drugs that aresafe are only marginally effective, while effective drugs haveunacceptable side effects with extensive monitoring of patientsrequired. Products under development continue to meet challenges relatedto side-effects (e.g., emesis side-effects characteristic of certainphosphodiesterase 4 inhibitors) and poor pharmacokinetic and metabolismparameters. There is a significant need for therapeutic agents thatachieve safe and effective treatment of inflammatory diseases such asasthma and allergy. The present invention provides these and relatedbenefits as described herein.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds according toformula (1) and pharmaceutically acceptable salts, solvates,stereoisomers and prodrugs thereof, in isolation or in mixtures,

-   -   wherein, independently at each occurrence:    -   R¹ and R² are selected from hydrogen, oxygen so as to form nitro        or oxime, amino, —SO₃—R, and organic groups having 1-30 carbons        and optionally containing 1-6 heteroatoms selected from        nitrogen, oxygen, phosphorous, silicon, and sulfur, where R² may        be a direct bond to numeral 3, or R¹ and R² may, together with        the N to which they are both bonded, form a heterocyclic        structure that may be part of an organic group having 1-30        carbons and optionally containing 1-6 heteroatoms selected from        nitrogen, oxygen and silicon; and where R¹ may be a 2, or 3 atom        chain to numeral 2 so that —N—R¹—forms part of a fused bicyclic        structure to ring A;    -   R³ and R⁴ are selected from direct bonds to 6 and 7 respectively        so as to form carbonyl groups, hydrogen, or a protecting group        such that R³ and/or R⁴ is part of hydroxyl or carbonyl        protecting group;    -   numerals 1 through 17 each represent a carbon, where carbons at        numerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independently        substituted with        -   (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),            —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein            n ranges from 1 to about 6; or        -   (b) two of the following, which are independently selected:            —X, —N(R¹)(R²), —R⁵ and —OR⁶;    -   and where carbons at numerals 5, 8, 9, 10, 13 and 14 may be        independently substituted with one of —X, —R⁵, —N(R¹)(R²) or        —OR⁶;    -   in addition to the —OR³ and —OR⁴ groups as shown, each of        carbons 6 and 7 may be independently substituted with one of —X,        —N(R¹)(R²), —R⁵ or —OR⁶;    -   each of rings A, B, C and D is independently fully saturated,        partially saturated or fully unsaturated;    -   R⁵ at each occurrence is independently selected from H, X, and        C₁₋₃₀ organic moiety that may optionally contain at least one        heteroatom selected from the group consisting of boron, halogen,        nitrogen, oxygen, silicon and sulfur; where two geminal R⁵        groups may together form a ring with the carbon atom to which        they are both bonded;    -   R⁶ is H or a protecting group such that —OR⁶ is a protected        hydroxyl group, where vicinal —OR⁶ groups may together form a        cyclic structure that protects vicinal hydroxyl groups, and        where geminal —OR⁶ groups may together form a cyclic structure        that protects a carbonyl group; and    -   X represents fluoride, chloride, bromide and iodide.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a steroid compound as set forth above, and apharmaceutically acceptable carrier, excipient or diluent.

In another aspect, the present invention provides a method of treatinginflammation comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatinginflammation prophylactically comprising administering to a subject inneed thereof a prophylactically-effective amount of a steroid compoundas set forth above.

In another aspect, the present invention provides a method of treatingasthma comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatingallergic disease including but not limited to dermal and ocularindications comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatingchronic obstructive pulmonary disease comprising administering to asubject in need thereof a therapeutically-effective amount of a steroidcompound as set forth above.

In another aspect, the present invention provides a method of treatingatopic dermatitis comprising administering to a subject in need thereofa therapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatingsolid tumours comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatingAIDS comprising administering to a subject in need thereof atherapeutically-effective amount of a steroid compound as set forthabove.

In another aspect, the present invention provides a method of treatingischemia reperfusion injury comprising administering to a subject inneed thereof a therapeutically-effective amount of a steroid compound asset forth above.

In another aspect, the present invention provides a method of treatingcardiac arrhythmias comprising administering to a subject in needthereof a therapeutically-effective amount of a steroid compound as setforth above.

These and related aspects of the present invention are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A and 1B depict summaries of synthetic transformations that maybe used to convert a 3-amino steroid into a 3-nitrogen steroid of thepresent invention.

FIGS. 2A, 2B and 2C are a set of bar graphs showing the effect ofcompound 89 (dose response, 4 doses qd, p.o.) on ovalbumin-inducedaccumulation of inflammatory cells in the lung lavage fluid obtainedfrom sensitized Brown Norway rats. FIG. 2A shows the accumulation ofeosinophils, FIG. 2B shows the accumulation of neutrophils, and FIG. 2Cshows the accumulation of lymphocytes.

FIGS. 3A, 3B and 3C are a set of bar graphs showing the effect ofcompound 28 (dose response, 4 doses qd, p.o.) on ovalbumin-inducedaccumulation of inflammatory cells in the lung lavage fluid obtainedfrom sensitized Brown Norway rats. FIG. 3A shows the accumulation ofeosinophils, FIG. 3B shows the accumulation of neutrophils, and FIG. 3Cshows the accumulation of lymphocytes.

FIG. 4 is a graph showing the effect of test compounds 28 and 89,administered orally once per day for 4 days prior to challenge, onallergen-induced changes in lung resistance in sensitized guinea pigs.

FIG. 5 is a graph showing the effect of test compounds 28 and 89,administered orally once per day for 4 days prior to challenge, onallergen-induced changes in lung elastance in sensitized guinea pigs.

FIG. 6 is a graph showing duration of anti-bronchospastic activity oftest compound 89, administered orally at 1 mg/kg once per day for 4 daysprior to challenge, on allergen-induced changes in lung resistance insensitized guinea pigs.

FIG. 7 is a graph showing duration of anti-bronchospastic activity oftest compound 89, administered orally at 1 mg/kg once per day for 4 daysprior to challenge, on allergen-induced changes in lung elastance insensitized guinea pigs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, compositions and methodsuseful in the treatment and/or prevention of various disease conditions.For example, in one aspect, the present invention provides a method oftreating and/or preventing an inflammatory disease. The method includesadministering to a subject in need thereof an effective amount of acompound of formula (1) or pharmaceutically acceptable salt, solvate,stereoisomer or prodrug thereof, or an effective amount of a compositioncontaining a compound of formula (1) or pharmaceutically acceptablesalt, solvate, stereoisomer or prodrug thereof.

Before describing the invention in further detail, certain definitionsas used herein are provided with the following definitions, and certainconventions used herein are also set forth.

A. Definition of Terms

As used herein, the following terms have the indicated meaning, unlessclearly indicated otherwise.

“Alkyl” is a monovalent, saturated or unsaturated, straight, branched orcyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In variousembodiments, the alkyl group has 1-20 carbon atoms, i.e., is a C1-C20(or C₁-C₂₀) group, or is a C1-C18 group, a C1-C12 group, a C1-C6 group,or a C1-C4 group. Independently, in various embodiments, the alkylgroup: has zero branches (ie., is a straight chain), one branch, twobranches, or more than two branches; is saturated; is unsaturated (wherean unsaturated alkyl group may have one double bond, two double bonds,more than two double bonds, and/or one triple bond, two triple bonds, ormore than two triple bonds); is, or includes, a cyclic structure; isacyclic. Exemplary alkyl groups include C₁alkyl (i.e., —CH₃ (methyl)),C₂alkyl (i.e., —CH₂CH₃ (ethyl), —CH═CH₂ (ethenyl) and —C≡CH (ethynyl))and C₃alkyl (i.e., —CH₂CH₂CH₃ (n-propyl), —CH(CH₃)₂ (i-propyl),—CH═CH—CH₃ (1-propenyl), —C≡C—CH₃ (1-propynyl), —CH₂—CH═CH₂(2-propenyl), —CH₂—C≡CH (2-propynyl), —C(CH₃)═CH₂ (1-methylethenyl), and—CH(CH₂)₂ (cyclopropyl)).

“Aryl” is a monovalent, aromatic, hydrocarbon, ring system. The ringsystem may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic,etc.). In various embodiments, the monocyclic aryl ring is C5-C10, orC5-C7, or C5-C6, where these carbon numbers refer to the number ofcarbon atoms that form the ring system. A C6 ring system, i.e., a phenylring, is a preferred aryl group. In various embodiments, the polycyclicring is a bicyclic aryl group, where preferred bicyclic aryl groups areC8-C12, or C9-C10. A naphthyl ring, which has 10 carbon atoms, is apreferred polycyclic aryl group.

“Heteroalkyl” is an alkyl group (as defined herein) wherein at least oneof the carbon atoms is replaced with a heteroatom. Preferred heteroatomsare nitrogen, oxygen, sulfur, and halogen. A heteroatom may, buttypically does not, have the same number of valence sites as carbon.Accordingly, when a carbon is replaced with a heteroatom, the number ofhydrogens bonded to the heteroatom may need to be increased or decreasedto match the number of valence sites of the heteroatom. For instance, ifcarbon (valence of four) is replaced with nitrogen (valence of three),then one of the hydrogens formerly attached to the replaced carbon mustbe deleted. Likewise, if carbon is replaced with halogen (valence ofone), then three (i.e., all) of the hydrogens formerly bonded to thereplaced carbon must be deleted.

“Heteroaryl” is a monovalent aromatic ring system containing carbon andat least one heteroatom in the ring. The heteroaryl group may, invarious embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may bemonocyclic or polycyclic, where the polycyclic ring may contained fused,spiro or bridged ring junctions. In one embodiment, the heteroaryl isselected from monocyclic and bicyclic. Monocyclic heteroaryl rings maycontain from about 5 to about 10 member atoms (carbon and heteroatoms),preferably from 5-7, and most preferably from 5-6 member atoms in thering. Bicyclic heteroaryl rings may contain from about 8-12 memberatoms, or 9-10 member atoms in the ring. The heteroaryl ring may beunsubstituted or substituted. In one embodiment, the heteroaryl ring isunsubstituted. In another embodiment, the heteroaryl ring issubstituted. Exemplary heteroaryl groups include benzofuran,benzothiophene, furan, imidazole, indole, isothiazole, oxazole,piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, quinoline, thiazole and thiophene.

“Heteroatom” is a halogen, nitrogen, oxygen, phosphorous, silicon orsulfur atom. Groups containing more than one heteroatom may containdifferent heteroatoms.

“Hydrocarbons” are chemical groups formed exclusively of hydrogen andcarbon; “Halocarbons” are chemical groups formed exclusively of halogenand carbon; and “Hydrohalocarbons” are chemical groups formedexclusively of hydrogen, halogen, and carbon.

“Organic groups” and “Organic moieties” are used synonymously, and referto stable structures having the indicated number and type of atoms.

“Pharmaceutically acceptable salt” and “salts thereof” in the compoundsof the present invention refers to acid addition salts and base additionsalts.

Acid addition salts refer to those salts formed from compounds of thepresent invention and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

Base addition salts refer to those salts formed from compounds of thepresent invention and inorganic bases such as sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum salts and the like. Suitable salts include the ammonium,potassium, sodium, calcium and magnesium salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

When any variable occurs more than one time in any constituent or incompounds of formula (1), its definition on each occurrence isindependent of its definition at every other occurrence. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds. The compounds useful in the methods andcompositions of the present invention, as well as the compounds of thepresent invention, may have asymmetric centers and occur as racemates,racemic mixtures and as individual diastereomers, or enantiomers withall isomeric forms being included in the present invention. A racemateor racemic mixture does not imply a 50:50 mixture of stereoisomers.

In another embodiment, the present invention provides pharmaceuticalcompositions containing a compound of formula (1) as set forth above, incombination with a pharmaceutically-acceptable carrier, diluent orexcipient. These compositions may be used for the treatment ofinflammation or other conditions as disclosed herein. These compositionsmay also be formed into a medicament, which may be used in the treatmentof, for example, inflammation.

These compositions are useful as, for example, assay standards,convenient means of making bulk shipments, or pharmaceuticalcompositions. An assayable amount of a compound of the invention is anamount which is readily measurable by standard assay procedures andtechniques as are well known and appreciated by those skilled in theart. Assayable amounts of a compound of the invention will generallyvary from about 0.001 wt % to about 100 wt % of the entire weight of thecomposition. Inert carriers include any material which does not degradeor otherwise covalently react with a compound of formula (1). Examplesof suitable inert carriers are water; aqueous buffers, such as thosewhich are generally useful in High Performance Liquid Chromatography(HPLC) analysis; organic solvents, such as acetonitrile, ethyl acetate,hexane and the like; and pharmaceutically acceptable carriers.

“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

Steroid compounds of the invention have at least four rings, commonlydesignated as A, B, C and D as shown below, where ring A may be fused toan additional ring:

B. Compounds

The present invention provides compounds according to formula (1) andpharmaceutically acceptable salts, solvates, stereoisomers and prodrugsthereof, in isolation or in mixtures,

-   -   wherein, independently at each occurrence:

R¹ and R² are selected from hydrogen, oxygen so as to form nitro oroxime, amino, —SO₃—R, and organic groups having 1-30 carbons andoptionally containing 1-6 heteroatoms selected from nitrogen, oxygen,phosphorous, silicon, and sulfur, where R² may be a direct bond tonumeral 3, or R¹ and R² may, together with the N to which they are bothbonded, form a heterocyclic structure that may be part of an organicgroup having 1-30 carbons and optionally containing 1-6 heteroatomsselected from nitrogen, oxygen and silicon, and where R¹ may be a 2, or3 atom chain to numeral 2 so that —N—R¹— forms part of a fused bicyclicstructure to ring A;

-   -   R³ and R⁴ are selected from direct bonds to 6 and 7 respectively        so as to form carbonyl groups, hydrogen, or a protecting group        such that R³ and/or R⁴ is part of hydroxyl or carbonyl        protecting group;    -   numerals 1 through 17 each represent a carbon, where carbons at        numerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independently        substituted with        -   (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),            —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein            n ranges from 1 to about 6; or        -   (b) two of the following, which are independently selected:            —X, —N(R¹)(R²), —R⁵ and —OR⁶;    -   and where carbons at numerals 5, 8, 9, 10, 13 and 14 may be        independently substituted with one of —X, —R⁵, —N(R¹)(R²) or        —OR⁶;    -   in addition to the —OR³ and —OR⁴ groups as shown, each of        carbons 6 and 7 may be independently substituted with one of —X,        —N(R¹)(R²), —R⁵ or —OR⁶;    -   each of rings A, B, C and D is independently fully saturated,        partially saturated or fully unsaturated;    -   R⁵ at each occurrence is independently selected from H, X, and        C₁₋₃₀ organic moiety that may optionally contain at least one        heteroatom selected from the group consisting of boron, halogen,        nitrogen, oxygen, silicon and sulfur; where two geminal R⁵        groups may together form a ring with the carbon atom to which        they are both bonded;    -   R⁶ is H or a protecting group such that —OR⁶ is a protected        hydroxyl group, where vicinal —OR⁶ groups may together form a        cyclic structure that protects vicinal hydroxyl groups, and        where geminal —OR⁶ groups may together form a cyclic structure        that protects a carbonyl group; and    -   X represents fluoride, chloride, bromide and iodide.

In one aspect of the invention, R¹ and R² are selected from hydrogen andorganic groups having 1-30 carbons and optionally containing 1-6heteroatoms selected from nitrogen, oxygen, phosphorous, silicon, andsulfur. Optionally, R² is a direct bond to numeral 3. In another aspect,R¹, R², and the N to which they are both bonded, form a heterocyclicstructure that may be part of an organic group having 1-30 carbons andoptionally containing 1-6 heteroatoms selected from nitrogen, oxygen andsilicon. In another aspect, R¹ is a 2, or 3 atom chain to numeral 2 sothat —N—R¹—forms part of a fused bicyclic structure to ring A, and the 2or 3 atoms are selected from C, N and O, so long as a stable structureresults. Optionally, in these and other aspects of the presentinvention, the organic group has 1-20 carbons, while in another optionalembodiment the organic group has 1-10 carbons.

In a preferred aspect of the invention, each of R¹ and R² is hydrogen.These steroids not only have desirable biological activity, they alsoserve as convenient precursor compounds to preparing other steroid ofthe invention wherein R¹ and/or R² is not hydrogen.

For example, in one embodiment, the invention provides a compound offormula (1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selected fromdirect bonds to 6 and 7 respectively so as to form carbonyl groups,hydrogen, or a protecting group such that R³ and/or R⁴ is part ofhydroxyl or carbonyl protecting group; and in addition to the —OR³ and—OR⁴ groups as shown, each of carbons 6 and 7 is substituted withhydrogen unless precluded because —OR³ or —OR⁴ represent a carbonylgroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵), —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and—(O(C(R⁵)(R⁵))_(n)O)— wherein n ranges from 1 to about 6; or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), —R⁵ and—OR⁶; each of rings A, B, C and D is independently fully saturated,partially saturated or fully unsaturated; R⁵ at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, oxygen, silicon and sulfur;where two geminal R⁵ groups may together form a ring with the carbonatom to which they are both bonded; R⁶ is H or a protecting group suchthat —OR⁶ is a protected hydroxyl group, where vicinal —OR⁶ groups maytogether form a cyclic structure that protects vicinal hydroxyl groups,and where geminal —OR⁶ groups may together form a cyclic structure thatprotects a carbonyl group; and X represents fluoride, chloride, bromideand iodide.

In another one embodiment, the invention provides a compound of formula(1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selected fromhydrogen and protecting groups such that R³ and/or R⁴ is part ofhydroxyl protecting group; carbons at numerals 1, 2, 4, 11, 12, 15 and16 are each substituted with two hydrogens; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), and—R⁵; each of rings A, B, C and D is independently fully saturated orpartially saturated; R⁵ at each occurrence is independently selectedfrom H, X, and C₁₋₃₀ hydrocarbons, halocarbons and halohydrocarbons; andX represents fluoride, chloride, bromide and iodide.

In another one embodiment, the invention provides a compound of formula(1) wherein: R¹ and R² are hydrogen; R³ and R⁴ are selected fromhydrogen and protecting groups such that R³ and/or R⁴ is part ofhydroxyl protecting group; carbons at numerals 1, 2, 4, 11, 12, 15 and16 are each substituted with two hydrogens; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of—R⁵; each of rings A, B, C and D is independently fully saturated orpartially saturated; and R⁵ at each occurrence is independently selectedfrom H and C₁₋₁₀ hydrocarbons.

Specific compounds of the present invention wherein R¹ and R² arehydrogen include:

In another aspect, the invention provides steroids having 3-nitrogensubstitution, where the 3-nitrogen is substituted with an organic group.For instance, the invention provides steroid compounds wherein R¹ isselected from —C(═O)—R⁷, —C(═O)NH—R⁷; and —SO₂—R⁷; wherein R⁷ isselected from alkyl, heteroalkyl, aryl and heteroaryl groups. In arelated embodiment, R¹ is hydrogen and R² is —CH₂—R⁷ wherein R⁷ isselected from alkyl, heteroalkyl, aryl and heteroaryl. In oneembodiment, R⁷ is selected from C₁₋₁₀hydrocarbyl. In another embodiment,—C(═O)—R⁷ comprises biotin. In another embodiment, R⁷ is selected fromalkyl-substituted phenyl; halogen-substituted phenyl; alkoxy-substitutedphenyl; aryloxy-substituted phenyl; and nitro-substituted phenyl.

In another aspect, (R¹)(R²)N— is a heterocycle, that is, the N of(R¹)(R²)N— may be part of a heterocyclic ring. Examples include:

In another aspect, either or both of R¹ and R² comprises a heterocyclicring or a carbocyclic ring. A preferred heterocyclic ring is

and a preferred carbocyclic ring is phenyl, which includes substitutedphenyl such as 3-methylphenyl; 4-hydroxyphenyl; and 4-sulfonamidephenyl.

In another aspect, R¹ may be a 2, or 3 atom chain to numeral 2 so that—N—R¹—forms part of a fused bicyclic structure to ring A. Thus, thepresent invention provides compounds of the formula shown below, where Zrepresents 2 or 3 atoms, selected from C, N and O. The ring including Zmay be saturated or unsaturated.

Examples of such fused ring compounds include:

In another aspect, R¹ is hydrogen and R² comprises a C₁₋₁₀hydrocarbyl.

In another aspect, R¹ is hydrogen and R² is heteroalkyl. Suitableheteroalkyl include, without limitation, C₁₋₁₀alkyl-W—C₁₋₁₀alkylene-wherein W is selected from O and NH; HO—C₁₋₁₀alkylene-; andHO—C₁₋₁₀alkylene-W—C₁₋₁₀alkylene- where W is selected from O and NH.

In another aspect, each of R¹ and R² is independently selected fromhydrogen and organic groups having 1-20 carbons and optionallycontaining 1-5 heteroatoms selected from nitrogen, oxygen, silicon, andsulfur.

In another aspect, each of R¹ and R² is independently selected fromhydrogen, R⁸, R⁹, R¹⁰, R¹¹ and R¹² where R⁸ is selected fromC₁₋₁₀alkyl,C₁₋₁₀heteroalkyl comprising 1, 2 or 3 heteroatoms, C₆₋₁₀aryland C₃₋₁₅heteroaryl comprising 1, 2 or 3 heteroatoms; R⁹ is selectedfrom (R⁸)_(r)—C₁₋₁₀alkylene, (R⁸)_(r)—C₁₋₁₀heteroalkylene comprising 1,2 or 3 heteroatoms, (R⁸)_(r)—C₆₋₁₀arylene and(R⁸)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹⁰ isselected from (R⁹)_(r)—C₁₋₁₀alkylene, (R⁹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁹)_(r)—C₆₋₁₀arylene, and(R⁹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹¹ isselected from (R¹⁰)_(r)—C₁₋₁₀alkylene, (R¹⁰)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹⁰)_(r)—C₆₋₁₀arylene, and(R¹⁰)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, R¹² isselected from (R¹¹)_(r)—C₁₋₁₀alkylene, (R¹¹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹¹)_(r)—C₆₋₁₀arylene, and(R¹¹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, and r isselected from 0, 1, 2, 3, 4 and 5, with the proviso that R¹ and R² mayjoin to a common atom so as to form a ring with the common atom.

In another aspect, the present invention provides steroid compounds ofthe structure shown above, wherein: R¹ and R² are independently selectedfrom hydrogen, R⁸ R⁹, R¹⁰, R¹¹ and R¹² where R⁸ is selected from alkyl,heteroalkyl, aryl and heteroaryl; R⁹ is selected from (R⁸)_(r)-alkylene,(R⁸)_(r)-heteroalkylene, (R⁸)_(r)-arylene and (R⁸)_(r)-heteroarylene;R¹⁰ is selected from (R⁹)_(r)-alkylene, (R⁹)_(r)-heteroalkylene,(R⁹)_(r)-arylene, and (R⁹)_(r)-heteroarylene; R¹¹ is selected from(R¹⁰)_(r)-alkylene, (R¹⁰)_(r)-heteroalkylene, (R¹⁰)_(r)-arylene, and(R¹⁰)_(r)-heteroarylene, R¹² is selected from (R¹¹)_(r)-alkylene,(R¹¹)_(r)-heteroalkylene, (R¹¹)_(r)-arylene, and(R¹¹)_(r)-heteroarylene, and r is selected from 0, 1, 2, 3, 4 and 5,with the proviso that R¹ and R² may join to a common atom so as to forma ring with the common atom; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of —R⁵; each ofrings A, B, C and D is independently fully saturated or partiallysaturated; and R⁵ at each occurrence is independently selected from Hand C₁₋₁₀ hydrocarbons.

For instance, R¹ and R² are selected from hydrogen, CH₃—, CH₃(CH₂)₂—,CH₃(CH₂)₄—, CH₃CO—, C₆H₅CO—(CH₃)₂CHSO₂—, C₆H₅SO₂—, C₆H₅NHCO—,CH₃(CH₂)₂NHCO—, CH₃(CH₂)₂NH(CH₂)₂—, (CH₃)₂N(CH₂)₂—, HOCH₂CH₂—,HOCH₂(CH₂)₄—, HOCH₂CH₂NHCH₂CH₂—, 3-(CH₃)C₆H₄—, 4-(HO)C₆H₄—,4-(H₂NSO₂)C₆H₄—, 4-((CH₃)₂CH)C₆H₄—CH₂—, 2-(F)C₆H₄—CH₂—,3-(CF₃)C₆H₄—CH₂—, 2-(CH₃O)C₆H₄—CH₂—, 4-(CF₃O)C₆H₄—CH₂—,3-(C₆H₅O)C₆H₄—CH₂—, 3-(NO₂)C₆H₄—CH₂—,

or R¹ and R² may join together with the nitrogen to which they are bothattached and form a heterocycle selected from:

Specific compounds of the present invention wherein R¹ is hydrogen butR² is not hydrogen include:

Thus, one set of preferred compounds of the invention have R¹ equal tohydrogen but R² is not equal to hydrogen.

In steroid compounds of the invention as disclosed above, in one aspecteach of R³ and R⁴ is hydrogen, i.e., the steroid has hydroxysubstitution at each of the carbons located at numerals 6 and 7. In arelated aspect, one or both of the hydroxy groups at carbons 6 and 7 arein a protected form, i.e., are bonded to a hydroxy protecting group.Such protecting groups are well known in the art, and are disclosed in,e.g., Greene and Wuts, “Protective Groups in Organic Synthesis”, JohnWiley & Sons, New York, N.Y. (1999). A suitable protecting group is aketal, so that the present invention provides compounds of thestructure:

As stated above, the present invention provides steroid compounds thatinclude compounds of defined stereochemistry. One such compound has thestereochemistry shown in the following structure for R³O—and R⁴O—:

As also stated above, the present invention provides salt forms of thesteroids of the present invention, preferably pharmaceuticallyacceptable salts. In one embodiment, —N(R¹)(R²) is in a salt form. Inother words, —N(R¹)(R²) is protonated so that the N carries a positivecharge. In such a case, the steroid compound of the present invention isan acid addition salt as defined herein. In a preferred aspect, thepresent invention provides hydrochloride salts of the steroid structuresshown above. In another preferred aspect, the present invention providesacetate salts of the steroid structures shown herein.

As also stated above, the present invention provides prodrugs of thespecific compounds shown by formula (1). In one aspect, the presentinvention is directed to a prodrug of any of the specific compoundsshown by formula (1). In another aspect, the present invention excludesprodrugs of the specific compounds shown by formula (1), i.e., in oneaspect the present invention is directed to compounds of formula (1) andpharmaceutically acceptable salts, solvates, stereoisomers but notprodrugs thereof, in isolation or in mixture.

In steroid compounds of the invention as set forth above, in a preferredembodiment, 17 is substituted with ═C(R⁵)(R⁵) and R⁵ is selected fromhydrogen, halogen, C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, and —CO₂—C₁₋₆alkyl. Inother preferred embodiment, 17 is substituted with C₁₋₆alkyl orC₁₋₆haloalkyl; or 17 is substituted with —OR⁶ or ═O, wherein R⁶ ishydrogen.

In steroid compounds of the invention as set forth herein, in apreferred embodiment, at least one of 10 and 13 is substituted withmethyl.

In steroid compounds of the invention as set forth herein, in apreferred embodiment, numerals 1 through 16 each represent a carbon,where carbons at numerals 1, 2, 4, 11, 12, 15 and 16 may beindependently substituted with: (a) one of: ═O, ═C(R⁵)(R⁵),═C═C(R⁵)(R⁵), —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)— whereinn ranges from 1 to about 6; or (b) two of the following, which areindependently selected: —X, —N(R¹)(R²), —R⁵ and —OR ⁶; and numeral 17represents a carbon substituted with: (a) one of: ═C(R^(5a))(R^(5a)),═C═C(R^(5a))(R^(5a)), and —C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)—wherein n ranges from 1 to about 6; or (b) two of the following, whichare independently selected: —X, —N(R¹)(R²), and —R^(5a); where R^(5a) ateach occurrence is independently selected from H, X, and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, silicon and sulfur;where two geminal R⁵ groups may together form a ring with the carbonatom to which they are both bonded. Optionally, R^(5a) at eachoccurrence is independently selected from C₁₋₃₀ hydrocarbon, C₁₋₃₀halocarbon, C₁₋₃₀ hydrohalocarbon, H, and X. In an alternative optionalembodiment, R at each occurrence is independently selected from C₁₋₁₀hydrocarbon, C₁₋₁₀ halocarbon, C₁₋₁₀ hydrohalocarbon, H, and X.Optionally, in each of these listed embodiment, the present inventionprovides a further embodiment wherein R¹ and R² are selected fromhydrogen, oxygen so as to form nitro or oxime, amino, —SO₃—R, andorganic groups having 1-30 carbons and optionally containing 1-6heteroatoms selected from oxygen, phosphorous, silicon, and sulfur,where R² may be a direct bond to numeral 3, or R¹ and R² may, togetherwith the N to which they are both bonded, form a heterocyclic structurethat may be part of an organic group having 1-30 carbons and optionallycontaining 1-6 heteroatoms selected from oxygen and silicon; or R¹ maybe a 2 or 3 atom chain to numeral 2 so that —N—R¹-forms part of a fusedbicyclic structure to ring A. Optionally, in each of these listedembodiments, the present invention provides a further embodiment whereincarbons at numerals 1, 2, 4, 11, 12, 15 and 16 are each substituted withtwo hydrogens unless said carbon is part of an unsaturated bond; carbonsat numerals 5, 8, 9 and 14 are each substituted with one hydrogen unlesssaid carbon is part of an unsaturated bond; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond. Optionally, in each ofthese listed embodiments, the present invention provides a furtherembodiment wherein carbons at numerals 1, 2, 4, 11, 12, 15 and 16 areeach substituted with two hydrogens; carbons at numerals 5, 8, 9 and 14are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond.

In steroid compounds of the invention as set forth herein, in apreferred embodiment, each of R¹ and R² is hydrogen; and/or each of R³and R⁴ is hydrogen; and/or the carbon at numeral 17 is substituted with(a) one of the following: C(R^(5a))(R^(5a)), ═C═C(R^(5a))(R^(5a)), and—C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)— wherein n ranges from 1 toabout 6; or (b) two of the following, which are independently selected:—X, —N(R¹)(R²), and —R^(5a); where R^(5a) at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, silicon and sulfur; where twogeminal R⁵ groups may together form a ring with the carbon atom to whichthey are both bonded.

In steroids of the present invention, unless otherwise indicated, eachof rings A, B, C and D is independently fully saturated, partiallysaturated or fully unsaturated. That is, hydrogens attached to any ofthe carbons at positions 1-17 may be omitted so as to allow unsaturationwithin the A, B, C and/or D ring. For example, when carbons at numerals5, 8, 9 and 14 are indicated as being substituted with one hydrogen, andit is also indicated that each of rings A, B, C and D is independentlyfully saturated, partially saturated or fully unsaturated, then any oneor more of the hydrogens attached to carbons at numerals 5, 8, 9 and 14may be omitted in order to allow unsaturation at the carbon atom.

The compounds of the present invention are intended as pharmaceuticalagents. Preferably, the molecular weight of a compound of the inventionis relatively small, that is, less than about 5,000 g/mol, typicallyless than 4,000 g/mol, more typically less than 3,000 g/mol, still moretypically less than 2,000 g/mol, yet more typically less than 1,000g/mol, where the minimum molecular weight of a compound of the inventionis about 300 g/mol, and each of these typical ranges is a separateembodiment of the present invention.

The steroid compounds of the present invention include pharmaceuticallyacceptable salts, solvates, stereoisomers and prodrugs of the3-nitrogen-6,7-dioxygenated steroid structures described above, inisolation or in mixtures with one another.

The steroid compounds of the invention may, and typically do, exist assolids, including crystalline solids which can be crystallized fromcommon solvents such as ethanol, N,N-dimethyl-formamide, water, or thelike. The crystallization process may, depending on the crystallizationconditions, provide various polymorphic structures. Typically, a morethermodynamically stable polymorph is advantageous to the commercialscale manufacture of a steroid compound of the invention, and is apreferred form of the compound.

Often, crystallizations produce a solvate of the steroid compound havingthe structure shown above. As used herein, the term “solvate” refers toan aggregate that comprises one or more 3-nitrogen-6,7-dioxygenatedsteroid compounds of the invention, with one or more molecules ofsolvent. The solvent may be water, in which case the solvate may be ahydrate. Alternatively, the solvent may be an organic solvent. Thus, thecompounds of the present invention may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,tetrahydrate and the like, as well as the corresponding solvated forms.The steroid compounds may be true solvates, while in other cases, thesteroid may merely retain adventitious water or be a mixture of waterplus some adventitious solvent.

As used herein, a “pharmaceutically acceptable solvate” refers to asolvate that retains the biological effectiveness and properties of thebiologically active 3-nitrogen-6,7-dioxygenated steroid compounds of theinvention. Examples of pharmaceutically acceptable solvates include, butare not limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine. It should be appreciated bythose skilled in the art that solvated forms are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention. Sykes, P. A., Guidebook to Mechanism in OrganicChemistry, 6th Ed (1986, John Wiley & Sons, N.Y.) is an exemplaryreference that describe solvates.

The inventive compounds may exist as single stereoisomers, racematesand/or mixtures of enantiomers and/or diastereomers. All such singlestereoisomers, racemates and mixtures thereof are intended to be withinthe scope of the present invention. In a preferred aspect, the inventivecompounds are used in optically pure form.

A “pharmaceutically acceptable prodrug” is intended to mean a compoundthat may be converted under physiological conditions or by solvolysis toa biologically active 3-nitrogen-6,7-dioxygenated steroid compound asdescribed above. Thus, the term “prodrug” refers to a metabolicprecursor of a steroid compound of the present invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject but is converted in vivo to an active3-nitrogen-6,7-dioxygenated steroid compound of the invention. Prodrugsare typically rapidly transformed in vivo to yield the parent compoundof the above formulae, for example, by hydrolysis in blood.

A discussion of prodrugs is provided in T. Higuchi and V. Stella,“Pro-drugs as Novel Delivery Systems,” Vol 14 of the A.C.S. SymposiumSeries, and in Bioreversible Carriers in Drug Design, ed. Edward B.Roche, American Pharmaceutical Association and Pergamon Press, 1987,both of which are incorporated herein by reference. A typical prodrug isa derivative of the steroid compounds of the invention which havechemically or metabolically cleavable groups and become, by solvolysisor under physiological conditions, the compounds of the invention whichare pharmaceutically active in vivo. The prodrug derivative form oftenoffers advantages of solubility, tissue compatibility, or delayedrelease in a mammalian organism (see, Bundgard, H., Design of Prodrugs,pp. 7-9, 21-24, Elsevier, Amsterdam 1985). A preferred prodrug is acompound having substitution at the 3-nitrogen atom of the steroids ofthe invention, where the substitution is cleaved in vivo to provide apharmaceutically active compound.

Steroids of the present invention having C3 nitrogen substitution andoxygen substitution at positions 6 and 7 have unexpected properties thatenhance the efficacies of these compounds. For instance, the steroid ofthe present invention have an excellent metabolic stability in S9fractions from human liver. For example, 100% of compounds 28, 89, 139,and 143 remains unchanged after 15 and even 30 minutes incubation withhuman S9 fractions. It was an unexpected finding that C3 nitrogensubstitutions significantly decrease glucuronidation of the molecules inplasma. In addition, steroids of the present invention having C3nitrogen substitutions such as compounds 28, 89 and 83 are highlysoluble in aqueous solution, demonstrating a solubility of >100 mg/ml inwater. Furthermore, the potency and pharmacokinetic profile of steroidsof the present invention with C3 nitrogen substitutions is highlysuitable for therapeutic application. Doses of <1.0 mg/kg once per dayreproducibly demonstrate significant anti-inflammatory activity in invivo inflammation models. In the rat, compounds 28 and 89 have anaverage half-life of 7.5 hours and an oral bioavailability of ˜100%,while in the monkey, the half-life averages 15 hours and oralbioavailability is 25-30%. The maximal concentration in plasma in bothspecies is predictable and linear.

C. Preparation of Compounds

The compounds according to the present invention can be prepared bymethods employing steps known to those skilled in the art or analogousto those steps. General methods for the reactions on steroids can befound in “Steroid Reactions”, C. Djerassi, Ed. Holden Day, SanFrancisco, Calif., 1963 and references cited therein. General syntheticmethods can be found in “Comprehensive Organic Transformations”, R. C.Larock, VCH Publishers, New York, N.Y., 1989 and references citedtherein. Additional literature references useful for the synthesis ofcompounds of the invention are as follows: T. Reichstein; C. H. Meystre,Helv. Chim. Acta, 1932, 22, 728; H. Westmijze; H. Kleyn; P. Vermeer; L.A. van Dijck, Tet. Lett. 1980, 21, 2665; K. Prezewowsky; R Wiechert,U.S. Pat. No. 3,682,983; P. Kaspar; H. Witzel, J. Steroid Biochem. 1985,23, 259; W. G. Dauben; T. Brookhart, J. Am. Chem. Soc. 1981, 103, 237;A. J. Manson et al., J. Med. Chem. 1963, 6, 1; R. O. Clinton et al, J.Am. Chem. Soc. 1961, 83, 1478; and J. A. Zderic et al. Chem. and Ind.1960, 1625.

In a preferred method, C3, C6, C7 and C17 polyoxygenated steroids areused as starting materials or intermediates. Methods to introduce C6 andC7 oxygens into commercially available starting materials are describedin U.S. Pat. No. 6,046,185. This U.S. Patent also discloses many ways inwhich substitution and preferred stereochemistry can be introduced intopositions C1, C2, C4, C5, C8, C9, C10, C11, C12, C13, C14, C15, C16 andC17. In the present invention, the C6 and C7 oxygens may be present ashydroxyls or as protected hydroxyls. The 6- and 7-hydroxyls can beprotected individually or they can together be part of a ring. Suitableprotecting groups are listed in Greene and Wuts, “Protective Groups inOrganic Synthesis”, John Wiley & Sons, New York, N.Y. (1999).

Referring to Scheme A, ketones of compound 2, or compounds analogousthereto, can be alkylated with a variety of alkylating groups to givesteroids of the invention having but not limited to, alkyl, cycloalkyl,aryl, and heteroaryl substitution. For example, alkylation of the17-ketone 2, with the anion of acetylene generates the17α-ethynyl-17β-hydroxyl intermediate 3. Reversal of the stereochemistryof the C17 substituents may be carried out by first forming themethylsulfonate followed by treatment with silver (I) nitrate intetrahydrofuran (THF) and water. Dehydration of compound 3 using POCl₃in 2,4-lutidine gives compound 4. Tetrabutylammonium fluoride in THFremoves the tert-butyldimethylsilyl (TBS) protecting group from the3-hydroxyl to give compound 5. Treatment of the 3α-hydroxyl compound 5with ZnN₆.2py, triphenylphosphine and diisopropyl azodicarboxylate(DIAD) in toluene gives the 3β-azido compound 6. The ZnN₆.2py isprepared by the reaction of Zn(NO₃)₂ and NaN₃ followed by treatment withpyridine according to the procedure of M. C. Viaud and P. Rollin inSynthesis 1990, 130. Lithium aluminum hydride reduction of the azide indiethyl ether (Et₂O) gives the amine 7. Treatment with HCl in THF andwater removes the acetonide group and forms the ammonium chloride salt8.

Referring to Scheme B, steroids of the invention having allenefunctionality may be prepared from intermediates analogous to compound3. Exemplary is the reaction of compound 3 with LiAlH₄ and AlCl₃ in THFto give the allene 9. Tetrabutylammonium fluoride in THF removes theprotecting group from the 3-hydroxyl to give compound 10. Treatment ofthe 3α-hydroxyl compound 10 with ZnN₆-2py, triphenylphosphine and DIADin toluene gives the 3β-azido compound 11. Lithium aluminum hydridereduction of the azide 11 in Et₂O gives the amine 12. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 13.

Referring to Scheme C, compounds of the invention having alkynylfunctionality may be prepared from allene intermediates. Exemplary isthe treatment of compound 9 with n-BuLi in THF giving the 17β-ethynylcompound 14. Tetrabutylammonium fluoride in THF removes the protectinggroup from the 3-hydroxyl to give compound 15. Treatment of the3α-hydroxyl compound 15 with ZnN₆.2py, triphenylphosphine and DIAD intoluene gives the 3β-azido compound 16. Lithium aluminum hydridereduction of the azide 16 in Et₂O gives the amine 17. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 18.

Referring to Scheme D, steroids of the invention having alkenylfunctionality may be prepared from alkyne intermediates. Exemplary isthe controlled hydrogenation of compound 14 using Pd—CaCO₃ as catalystto give the alkene 19. Tetrabutylammonium fluoride in THF removes theprotecting group from the 3-hydroxyl to give compound 20. Treatment ofthe 3α-hydroxyl compound 20 with ZnN₆-2py, triphenylphosphine and DIADin toluene gives the 3β-azido compound 21. Lithium aluminum hydridereduction of the azide 21 in Et₂O gives the amine 22. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 23.

Compound 2 can be used in a multitude of olefination reactions,including Wittig-type-reactions to provide compounds of the inventionhaving an exocyclic olefin at C17. For example, as illustrated in SchemeE, compound 2 may be treated with ethyltriphenylphosphonium bromide andpotassium tert-butoxide (KO^(t)Bu) to provide compound 24 havingR₁=methyl and R₂=hydrogen. Tetrabutylammonium fluoride in THF removesthe protecting group from the 3-hydroxyl to give compound 25. Treatmentof the 3α-hydroxyl compound 25 with ZnN₆.2py, triphenylphosphine andDIAD in toluene gives the 3β-azido compound 26. Lithium aluminum hydridereduction of the azide 26 in Et₂O gives the amine 27. Treatment with HClin THF and water removes the acetonide group and forms the ammoniumchloride salt 28 having R₁=methyl and R₂=hydrogen.

In analogy to the synthesis shown in Scheme E, ketones such as compound2 may be reacted with other Wittig-type reagents such as, but notlimited to, methyl-, propyl-, butyl-, pentyl-, orhexyltriphenylphosphonium bromide to give steroids of the inventionanalogous to compound 28 having R₂=hydrogen and R₁=hydrogen, ethyl,propyl, butyl or pentyl.

Steroids of the invention can contain exocyclic double bonds of E and/orZ geometry. For example, as illustrated in Scheme F, the Z-olefin 24 incyclohexane may be treated with UV light in the presence ofdiphenyldisulfide resulting in isomerization to the E-olefin 29.Tetrabutylammonium fluoride in THF removes the protecting group from the3-hydroxyl to give compound 30. Treatment of the 3α-hydroxyl compound 30with ZnN₆.2py, triphenylphosphine and DIAD in toluene gives the 3β-azidocompound 31. Lithium aluminum hydride reduction of the azide 31 in Et₂Ogives the amine 32. Treatment with HCl in THF and water removes theacetonide group and forms the ammonium chloride salt 33.

A multitude of steroids of the invention having functionalizedsidechains can be prepared using methods such as Lewis acid promotedcouplings to alkehydes and Michael acceptors. For example, asillustrated in Scheme G, compound 24 may be reacted with methylpropiolate in the presence of diethylaluminum chloride to give compound34. The double bonds may be hydrogenated using a catalyst such asplatinum to give compound 35. Tetrabutylammonium fluoride in THF removesthe protecting group from the 3-hydroxyl to give compound 36. Treatmentof the 3α-hydroxyl compound 36 with ZnN₆.2py, triphenylphosphine andDIAD in toluene gives the 3β-azido compound 37. Hydrogenation of theazide 37 using a palladium catalyst gives the amine 38. Treatment withHCl in THF and water removes the acetonide group and forms the ammoniumchloride salt 39.

With a 3-amino steroid, such as prepared by any of the above SchemesA-G, a large variety of secondary and tertiary amine compounds of theinvention can be prepared. FIGS. 1A and 1B outline several syntheticpathways that may be employed to prepare 3-amino compounds of thepresent invention. For instance, reductive amination methods may be usedto couple primary (see FIG. 1A) and secondary (see FIG. 1B) amines withaldehydes (RC(═O)H) and ketones (RC(═O)R′). Although not shown in eitherof FIG. 1A or 1B, compounds having two aldehyde groups, i.e.,dialdehydes of the general formula HC(═O)—R—C(═O)H, may be reacted with3-amino steroids to provide steroids having heterocyclic structures atthe 3-position. In addition (or alternatively), reductive aminationmethods may be used to couple 3-keto steroids with heterocyclicsecondary amines. By these approaches, the present invention providescompounds wherein R¹ and R² may, together with the N to which they areboth bonded, form a heterocyclic structure that may be part of anorganic group having 1-30 carbons and optionally containing 1-6heteroatoms selected from nitrogen, oxygen and silicon. Commercialsources and reference to the chemical literature provides one ofordinary skill in the art with access to a multitude of aldehydes(including dialdehyes) and ketones that may be used to prepare steroidcompounds of the present invention. Reductive amination methods aredescribed in, for example Synthesis 1975, 135; J. Am. Chem. Soc. 1971,93, 2897; M. Freifelder in “Catalytic Hydrogenation in OrganicSynthesis” J. Wiley & Sons 1978, Ch. 10; Russ. Chem. Rev. 1980, 49, 14,and references cited therein. See also, J. Chem. Soc. Perkin Trans 11998, 2527; and Synlett 1999, 1781, as well as references cited therein.

Primary (see FIG. 1A) and secondary (see FIG. 1B) amines can be coupledto aryl compounds (ArX) to generate a variety of aryl substituted aminecompounds of the invention. Commercial sources and reference to thechemical literature provides one of ordinary skill in the art withaccess to a multitude of aryl compounds that may be used to preparesteroid compounds of the present invention. Examples of methods for theamination of aryl compounds can be found in J. Org. Chem. 2000, 65, 1158and in the review Angew. Chem. Int. Ed. 1998, 37, 2046 and referencescited therein.

Methods to react primary (see FIG. 1A) and secondary (see FIG. 1B)amines with acyl chlorides (RC(═O)Cl) and sulfonyl chlorides (RSO₂Cl) togenerate amide and sulfonamide compounds of the invention, respectively,are well known to those skilled in the art of organic chemistry, in thecontext of other amine compounds, and these same techniques may beapplied to the amine compounds of the present invention. Commercialsources and reference to the chemical literature provides one ofordinary skill in the art with access to a multitude of acyl chloridesand sulfonyl chlorides that may be used to prepare steroid compounds ofthe present invention.

Methods to react primary (see FIG. 1A) and secondary (see FIG. 1B)amines with isocyanates (RN═C═O) and isothiocyanates (RN═C═S) togenerate ureas and thioureas, respectively, are also well known to thoseskilled in the art of organic chemistry, in the context of other aminecompounds, and these same techniques may be applied to the aminecompounds of the present invention. Commercial sources and reference tothe chemical literature provides one of ordinary skill in the art withaccess to a multitude of isocyanates and isothiocyanates that may beused to prepare steroid compounds of the present invention. The reviewarticle Russ. Chem. Rev. 1985, 54, 249 and references cited thereindescribes examples of the variety of substituted ureas and thioureasthat can be encompassed by the invention.

Thus, by using appropriately selected aldehydes, ketones, arylcompounds, acyl chlorides, sulfonyl chlorides, isocyanates and/orisothiocyanates, one of ordinary skill in the art may prepare steroidcompounds wherein R¹ and R² are selected from hydrogen and organicgroups having 1-30 carbons and optionally containing 1-6 heteroatomsselected from nitrogen, oxygen, phosphorous, silicon, and sulfur.

Steroids of the invention may have fused heterocycles such as, but notlimited to, pyrazole, isoxazole and pyrimidine. As illustrated in SchemeH, compound 43 is an example of a fused pyrazole of the invention, forwhich the synthesis of the starting material compound 40 is described inU.S. Pat. No. 6,046,185. Treatment of compound 40 with ethyl formate inpyridine in the presence of NaOMe gives the hydroxymethyleneintermediate 41. Reaction of compound 41 with hydrazine hydrate in EtOHforms the pyrazole compound 42, which upon treatment withtetrabutylammonium fluoride in THF gives the compound 43.

As illustrated in Scheme I, intermediates such as compound 41 may beconverted into isoxazoles of which compound 44 is exemplary. Treatmentof the hydroxymethylene intermediate 41 with ammonium hydroxide inpyridine followed by deprotection of the 6- and 7-hydroxyls usingtetrabutylammonium fluoride in THF gives the isoxazole 44.

As illustrated in Scheme J, intermediates such as compound 41 may beconverted into pyrimidines of which compound 44a is exemplary. Treatmentof the hydroxymethylene intermediate 41 with benzamidine hydrochlorideand potassium hydroxide in ethanol followed by deprotection of the 6-and 7-hydroxyls using tetrabutylammonium fluoride in THF gives thepyrimidine 44a.

Thus, by proceeding through compounds having keto substitution as carbon3 and ═CHOH substitution at carbon 2, the present invention providesaccess to a multitude of compounds wherein R¹ may be a 2 or 3 atom chainto numeral 2 so that —N—R¹— forms part of a fused bicyclic structure toring A.

Reaction of 3-keto steroids with hydroxylamine and pyridine may beemployed to produce steroid oximes of the invention. A steroid oxime hasR² as a direct bond to numeral 3, thus providing a double bond betweenthe carbon at numeral 3 and the N, and R¹ is OH. Primary amines may beoxidized to nitro compounds by, for instance, dimethyldioxirane. Thus,R¹ and R² may be oxygen. Methods to generate the nitro functionality aredescribed in J. Org. Chem. 1989, 54, 5783. Reaction of 3-ketones withdimethylhydrazine gives N,N-dimethylhydrazone steroids of the inventionin which R² is a direct bond to numeral 3 and R¹ is NMe₂. Treatment ofdimethylhydrazone steroids with hydrazine generates hydrazone steroidsof the invention. Description of the methods to generateN,N-dimethylhydrazones and hydrazones can be found in J. Org. Chem.1966, 31, 677. Primary amines may also be reacted by methodology knownin the art with sulfonic/sulfuric acids and esters to provide sulfamatecompounds, i.e., steroids wherein numeral 3 is bonded to —N—SO₃—R and Ris H or an organic group having 1-30 carbons and optionally containing1-6 heteroatoms selected from nigrogen, oxygen, phosphorous, silicon,and sulfur.

D. Pharmaceutical Compositions

The present invention provides a pharmaceutical or veterinarycomposition (hereinafter, simply referred to as a pharmaceuticalcomposition) containing a compound of formula (1) as described above, inadmixture with a pharmaceutically acceptable carrier. The inventionfurther provides a composition, preferably a pharmaceutical composition,containing an effective amount of a compound as described above, inassociation with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be in anyform which allows for the composition to be administered to a patient.For example, the composition may be in the form of a solid, liquid orgas (aerosol). Typical routes of administration include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,ocular, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Pharmaceutical composition of theinvention are formulated so as to allow the active ingredients containedtherein to be bioavailable upon administration of the composition to apatient. Compositions that will be administered to a patient take theform of one or more dosage units, where for example, a tablet may be asingle dosage unit, and a container of a compound of formula (1) inaerosol form may hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions should bepharmaceutically pure and non-toxic in the amounts used. It will beevident to those of ordinary skill in the art that the optimal dosage ofthe active ingredient(s) in the pharmaceutical composition will dependon a variety of factors. Relevant factors include, without limitation,the type of subject (e.g., human), the particular form of the activeingredient, the manner of administration and the composition employed.

In general, the pharmaceutical composition includes an (where “a” and“an” refers here, and throughout this specification, as one or more)active compound of formula (1) as described herein, in admixture withone or more carriers. The carrier(s) may be particulate, so that thecompositions are, for example, in tablet or powder form. The carrier(s)may be liquid, with the compositions being, for example, an oral syrupor injectable liquid. In addition, the carrier(s) may be gaseous, so asto provide an aerosol composition useful in, e.g., inhalatoryadministration.

When intended for oral administration, the composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the composition may beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition willtypically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following adjuvants may be present: binderssuch as carboxymethylcellulose, ethyl cellulose, microcrystallinecellulose, or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin, a flavoring agent such as peppermint,methyl salicylate or orange flavoring, and a coloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it may contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, cyclodextrin, propylene glycol or othersolvents; antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of a compound of formula (1)such that a suitable dosage will be obtained. Typically, this amount isat least 0.01% of a compound of the invention in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1% and about 80% of the weight of the composition. Preferredoral compositions contain between about 4% and about 50% of the activecompound of formula (1). Preferred compositions and preparationsaccording to the present invention are prepared so that a parenteraldosage unit contains between 0.01% to 2% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of formula (1) of from about 0.1% to about10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The composition may include various materials which modify the physicalform of a solid or liquid dosage unit. For example, the composition mayinclude materials that form a coating shell around the activeingredients. The materials which form the coating shell are typicallyinert, and may be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients may beencased in a gelatin capsule.

The composition in solid or liquid form may include an agent which bindsto the active component(s) and thereby assists in the delivery of theactive components. Suitable agents which may act in this capacityinclude a monoclonal or polyclonal antibody, a protein or a liposome.

The pharmaceutical composition of the present invention may consist ofgaseous dosage units, e.g., it may be in the form of an aerosol. Theterm aerosol is used to denote a variety of systems ranging from thoseof colloidal nature to systems consisting of pressurized packages.Delivery may be by a liquefied or compressed gas or by a suitable pumpsystem which dispenses the active ingredients. Aerosols of compounds ofthe invention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, spacers and the like, which together may form a kit.Preferred aerosols may be determined by one skilled in the art, withoutundue experimentation.

Whether in solid, liquid or gaseous form, the pharmaceutical compositionof the present invention may contain one or more known pharmacologicalagents used in the treatment of inflammation (including asthma, allergy,rheumatoid arthritis, multiple sclerosis, etc.), proliferative disorders(cancers), diseases treatable through the regulation of calcium(including hypertension, cardiac arrhythmias, etc.), and Acquired ImmuneDeficiency Syndrome (AIDS).

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art.

A composition intended to be administered by injection can be preparedby combining the compound of formula (1) with water so as to form asolution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of formula (1) so as tofacilitate dissolution or homogeneous suspension of the active compoundin the aqueous delivery system.

E. Biological Activity

The compounds disclosed herein of formula 1, or compositions comprisingone of more of these compounds and a pharmaceutically acceptablecarrier, diluent or excipient, may be used in a method for treating orpreventing an inflammatory condition or disease in a patient, where themethod comprises administering to the patient in need thereof an amountof a compound or composition according to the present invention, wherethe amount is effective to treat or prevent the inflammatory conditionor disease of the patient.

The inflammatory condition or disease may involve respiratoryinflammation (e.g., wherein the respiratory disease is asthma, orwherein the respiratory disease is chronic obstructive pulmonarydisease; or wherein the respiratory disease is emphysema); theinflammatory condition may be an autoimmune condition or disease; theinflammatory condition or disease may be lupus erythematosus disease;the inflammatory condition or disease may involve acute or chronicinflammation of bone and/or cartilage compartments of joints; theinflammatory condition or disease may be an arthritis selected fromrheumatoid arthritis, gouty arthritis or juvenile rheumatoid arthritis;the inflammatory condition or disease may be a central nervous systemdisease; the condition or disease may be associated with leukocyteinfiltration; the condition or disease may be associated with edema; thecondition or disease may be associated with ischemia reperfusion injury;the condition or disease may be associated with elevated levels ofinflammatory cytokines (e.g., wherein the inflammatory cytokine isinterleukin (IL)-4, or wherein the inflammatory cytokine is IL-5, orwherein the inflammatory cytokine is IL-10, or wherein the inflammatorycytokine is IL-13, or wherein the inflammatory cytokine is IL-9, orwherein the inflammatory cytokine is IL-1, or wherein the inflammatorycytokine is IL-2, or wherein the inflammatory cytokine is IL-6, orwherein the inflammatory cytokine is IL-18, or wherein the inflammatorycytokine is IL-3, or wherein the inflammatory cytokine is IL-8, orwherein the inflammatory cytokine is IL-12, or wherein the inflammatorycytokine is TNF-α, or wherein the inflammatory cytokine is TGF-β, orwherein the inflammatory cytokine is GM-CSF, or wherein the inflammatorycytokine is IFN-γ, or wherein the inflammatory cytokine is LTB4, orwherein the inflammatory cytokine is a member of the cysteinylleukotriene family, or wherein the inflammatory cytokine is regulated onactivation normal T cell expressed and secreted (RANTES), or wherein theinflammatory cytokine is eotaxin-1, 2, or 3, or wherein the inflammatorycytokine is macrophage inflammatory protein (MIP)-1α, or wherein theinflammatory cytokine is monocyte chemoattractant protein-1, 2, 3, or4,); the condition or disease may be associated with altered levels ofinflammatory adhesion molecules (e.g., wherein the adhesion molecule isvascular cell adhesion molecule (VCAM-1 or 2), wherein the adhesionmolecule is intercellular adhesion molecule (ICAM-1 or 2), wherein theadhesion molecule is very late antigen-4 (VLA-4), wherein the adhesionmolecule is leukocyte function associated antigen-1 (LFA-1); wherein theadhesion molecule is a selectin); the inflammatory condition or diseasemay be multiple sclerosis; the inflammatory condition or disease may bepulmonary sarcadosis; the inflammatory condition or disease may beocular inflammation or allergy; the inflammatory condition or diseasemay be allergic rhinitis; the inflammatory condition or disease may bean inflammatory bowel disease (e.g., Crohn's disease or ulcerativecolitis); the inflammatory condition or disease may be an inflammatorycutaneous disease (e.g., psoriasis or dermatitis); the inflammatorycondition or disease may be graft vs host disease; the inflammatorycondition or disease may be vascular (e.g., vasculitis); theinflammatory condition or disease may be an atherosclerotic disease.

Furthermore, the present invention provides a method for treating orpreventing a disease or condition in a patient, where the disease orcondition is associated with pathological conditions that involveleukocyte infiltration, the method comprising administering to a patientin need thereof an amount of a compound or a composition of the presentinvention, wherein the amount is effective to treat or prevent a diseaseor condition associated with pathological conditions that involveleukocyte infiltration.

Furthermore, the present invention provides a method of treating orpreventing asthma in a patient, comprising administering to a patient inneed thereof an amount of a compound or composition of the presentinvention, where the amount is effective to treat or prevent asthma inthe patient.

Furthermore, the present invention provides a method of treating orpreventing allergy in a patient, comprising administering to a patientin need thereof an amount of a compound or composition of the presentinvention, where the amount is effective to treat or prevent allergy inthe patient.

In a method of the present invention, a compound of formula (1), or acomposition comprising one or more compounds of formula (1) and apharmaceutically acceptable carrier, diluent or excipient, may, althoughneed not, achieve one or more of the following desired results in thesubject to whom has been administered a compound of formula (1) asdefined above, or a composition containing one of these compounds and apharmaceutically acceptable carrier, diluent or excipient:

-   -   1. Inhibition of leukocyte infiltration (e.g., neutrophils,        eosinophils, etc.)    -   2. Inhibition of leukocyte activation    -   3. Alteration of lymphocyte ratio (e.g., TH1 vs TH2 cells)    -   4. Inhibition of leukocyte chemotaxis;    -   5. Inhibition of TNF-α production and/or release;    -   6. Inhibition of chemokine production and/or release (e.g.,        eotaxin, etc.);    -   7. Inhibition of adhesion molecule production, release and/or        function (e.g., VCAM, VLA-4, etc.);    -   8. Inhibition of edema;    -   9. Inhibition of interleukin cytokine production and/or release        (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-8, IL-9, IL-10,        IL-12, IL-13, IL-18,);    -   10. Inhibition of inflammatory mediator release (e.g.,        leukotrienes, tryptase, adenosine etc.);    -   11. Inhibition of histamine release;    -   12. Inhibition of parameters of asthma; and    -   13. Inhibition of parameters of allergy.

The compounds disclosed herein of formula 1 (i.e., compounds of formulae(1), or compounds of the present invention), or compositions comprisingone of more of these compounds and a pharmaceutically acceptablecarrier, diluent or excipient, may be used in a method for treating orpreventing a proliferative disorder in a patient, where the methodcomprises administering to the patient in need thereof an amount of acompound or composition according to the present invention, where theamount is effective to treat or prevent the proliferative disorder ofthe patient. As used herein, proliferative disorders includes, withoutlimitation, all leukemias and solid tumors that are susceptible toundergoing differentiation or apoptosis upon interruption of their cellcycle.

The compounds disclosed herein of formula 1 (i.e., compounds of formulae(1), or compounds of the present invention), or compositions comprisingone or more of these compounds and a pharmaceutically acceptablecarrier, diluent or excipient, may be used in a method for treating orpreventing diseases treatable through regulation of calcium in apatient, where the method comprises administering to the patient in needthereof an amount of a compound or composition according to the presentinvention, where the amount is effective to treat or prevent the diseaseof the patient. As used herein, diseases treatable through regulation ofcalcium includes, without limitation, cardiac arrhythmia, atrialfibrillation, acute coronary syndromes, hypertension, ischemiareperfusion injury, stroke, epilepsy, demyelinating diseases such asmultiple sclerosis, pain, status epilepticus, artherosclerosis, anddiabetes.

The compounds disclosed herein of formula 1 (i.e., compounds of formulae(1), or compounds of the present invention), or compositions comprisingone or more of these compounds and a pharmaceutically acceptablecarrier, diluent or excipient, may be used in a method for treating orpreventing Acquired Immunodeficiency Syndromes (AIDS) in a patient,where the method comprises administering to the patient in need thereofan amount of a compound or composition according to the presentinvention, where the amount is effective to treat or prevent theAcquired Immunodeficiency Syndromes of the patient. As used herein,Acquired Immunodeficiency Syndromes through infection with humanimmunodeficiency virus type 1 includes, without limitation, associatedcomplications such as Acquired Immunodeficiency Syndrome DementiaComplex, and neuro-Acquired Immunodeficiency Syndromes.

Thus, the inventive method may be used to treat inflammation, includingboth acute and chronic inflammation, as well as certain proliferativedisorders (cancers), diseases treatable through regulation of calcium,and AIDS. As used herein, inflammation includes, without limitation,ankylosing spondylitis, arthritis (where this term encompasses over 100kinds of rheumatic diseases), asthma, chronic obstructive pulmonarydisease, allergy, allergic rhinitis, Crohn's disease, fibromyalgiasyndrome, gout, inflammations of the brain (including multiplesclerosis, AIDS dementia, Lyme encephalopathy, herpes encephalitis,Creutzfeld-Jakob disease, and cerebral toxoplasmosis), emphysema,inflammatory bowel disease, irritable bowel syndrome,ischemia-reperfusion injury, atopic dermatitis, juvenile erythematosuspulmonary sarcoidosis, Kawasaki disease, osteoarthritis, pelvicinflammatory disease, psoriatic arthritis (psoriasis), rheumatoidarthritis, psoriasis, tissue/organ transplant, graft vs host disease;scleroderma, spondyloarthropathies, systemic lupus erythematosus,pulmonary sarcoidosis, vasculitis, artherosclerosis, cardiomyopathy,autoimmune myocarditis, and ulcerative colitis.

The inventive method provides for administering a therapeuticallyeffective amount of a compound of formula (1), including salts,compositions etc. thereof. As used herein, the actual amount encompassedby the term “therapeutically effective amount” will depend on the routeof administration, the type of warm-blooded animal being treated, andthe physical characteristics of the specific warm-blooded animal underconsideration. These factors and their relationship to determining thisamount are well known to skilled practitioners in the medical arts. Thisamount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors that those skilled in themedical arts will recognize.

An effective amount of a compound or composition of the presentinvention will be sufficient to treat inflammation, proliferativediseases, diseases treatable by regulation of calcium, or AIDS, in awarm-blooded animal, such as a human. Methods of administering effectiveamounts of anti-inflammatory agents are well known in the art andinclude the administration of inhalation, oral or parenteral forms. Suchdosage forms include, but are not limited to, parenteral solutions,tablets, capsules, sustained release implants and transdermal deliverysystems; or inhalation dosage systems employing dry powder inhalers orpressurized multi-dose inhalation devices.

The dosage amount and frequency are selected to create an effectivelevel of the agent without harmful effects. It will generally range froma dosage of about 0.001 to 100 mg/Kg/day, and typically from about 0.01to 10 mg/Kg/day where administered orally or intravenously. Also, thedosage range will be typically from about 0.0001 to 10 mg/Kg/day whereadministered intranasally or by inhalation.

The compounds of formula (1) including the compounds used in the methodsand compositions set forth above, may be prepared according to theSchemes set forth in the following examples. The following examples areoffered by way of illustration and not by way of limitation.

Unless otherwise stated, flash chromatography and column chromatographymay be accomplished using Merck silica gel 60 (230-400 mesh). Flashchromatography may be carried out according to the procedure set forthin: “Purification of Laboratory Chemicals”, 3rd. edition,Butterworth-Heinemann Ltd., Oxford (1988), Eds. D. D. Perrin and W. L.F. Armarego, page 23. Column chromatography refers to the processwhereby the flow rate of eluent through a packing material is determinedby gravity. In all cases flash chromatography and radial chromatographymay be used interchangeably. Radial chromatography is performed usingsilica gel on a Chromatotron Model # 7924T (Harrison Research, PaloAlto, Calif.). Unless otherwise stated, quoted R_(f) values are obtainedby thin layer chromatography using Silica Gel 60 F₂₅₄ (Merck KGaA,64271, Darmstadt, Germany). Brine refers to a saturated solution ofsodium chloride.

Also, unless otherwise stated, chemical reactants and reagents wereobtained from standard chemical supply houses, such as Aldrich(Milwaukee, Wis.; www.aldrich.sial.com); EM Industries, Inc. (Hawthorne,N.Y.; www.emscience.com); Fisher Scientific Co. (Hampton, N.H.;www.fischer1.com); and Lancaster Synthesis, Inc. (Windham, N.H.;www.lancaster.co.uk). Sulfo-NHS-biotin was obtained from Pierce(Rockford, Ill., www.piercenet.com). MP-TsOH resin, PS-DIEA resin,PS-Trisamine resin and PS-Benzaldehyde resin were obtained from ArgonautTechnologies (San Carlos, Calif., www.argotech.com). Gases were obtainedfrom Praxair (Vancouver, B.C.). Cell lines, unless otherwise stated,where obtained from public or commercial sources, e.g., American TissueCulture Collection (ATCC, Rockville, Md.).

SYNTHESIS EXAMPLES Example 1 3-Amino-6,7-Dihydroxy-17-Ethylidene Steroid

Compound 49, a representative compound of the invention, is preparedaccording to Scheme 1. Any number of compounds related to compound 49could be produced using similar methodology. The starting materialcompound 45 can be prepared according to the methodology described inU.S. Pat. No. 6,046,185. Olefination of the ketone 45 is accomplishedusing ethyltriphenylphosphonium bromide and KO^(t)Bu in toluene.Treatment of the 3β-hydroxyl compound 46 with ZnN₆.2py,triphenylphosphine and DIAD in toluene produced the 3α-azido compound47. Lithium aluminum hydride reduction of the azide in Et₂O provided theamine 48. Treatment with HCl in THF and water removes the acetonidegroup and forms the ammonium chloride salt 49.

Synthesis of Compound 46

A solution of KO^(t)Bu (0.24 g, 2.0 mmol), EtPPh₃Br (0.75 g, 2.0 mmol)and toluene (2.5 ml) was stirred at room temperature under argon. After1 hour the deep red solution was cooled in ice and the ketone 45 (184mg, 0.508 mmol) was added and the resulting solution was allowed to warmto room temperature. After stirring overnight the reaction was quenchedwith 10 ml of water, diluted with 60 ml of ethyl acetate (EtOAc),separated and washed with 2×10 ml of brine, dried over MgSO₄, filteredand concentrated. Purification by column chromatography eluting with 1:1EtOAc/hexanes afforded 171 mg (90%) of compound 46 as a colorless film.

Synthesis of Compound 47

DIAD (0.44 ml, 2.14 mmol) was added dropwise over 10 minutes to a roomtemperature solution of the 3β-hydroxy compound 46 (400 mg, 1.07 mmol),ZnN₆.2py (246 mg, 0.80 mmol), Ph₃P (560 mg, 2.14 mmol) and toluene (10.7ml) under argon. After 4 hours the reaction mixture was loaded onto acolumn of silica gel packed in 10% ethyl acetate/hexanes and eluted with20% ethyl acetate/hexanes to afford 422 mg (99%) of compound 47 as awhite solid.

Synthesis of Compound 48

Lithium aluminum hydride (42 mg, 1.04 mmol) was added to an ice cooledsolution of the azide 47 (415 mg, 1.04 mmol) in 5.2 ml of Et₂O underargon. The reaction was allowed to warm to room temperature. After 2hours the solution was cooled in ice, diluted with 25 ml of diethylether and slowly quenched with 2 ml of saturated Na₂SO₄ solution. After10 minutes a white precipitate had formed and the solution was dilutedwith 50 ml of ethyl acetate, washed with 3×10 ml of brine, dried overMgSO₄, filtered and concentrated. The crude material was purified usinga column of silica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washingwith 5% MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, elutedwith 5% MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give a whitefoam which was shown by ¹H nmr to contain a trace of Et₃N. The materialwas taken up in 50 ml of hexanes, washed with 2×20 ml of brine, driedover MgSO₄, filtered and concentrated to give 322 mg (83%) of compound48 as a white foam.

Synthesis of Compound 49

A solution of the 3α-amino compound 48 (317 mg, 0.850 mmol), 4 M HCl indioxane (255 μl, 1.02 mmol), THF (13.6 ml) and water (3.4 ml) wasstirred at room temperature overnight. The solution was concentrated todryness, triturated with 3×10 ml portions of acetone, evaporating offthe acetone after each trituration. Concentration gave 301 mg (96%) ofcompound 49 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 334.16; C₂₁H₃₆NO₂.

Example 2 3-Amino-6,7-Dihydroxy-17-Methylidene Steroid

A further example of alkenes related to compound 49 is shown in Scheme2. Olefination of the ketone 45 using methyltriphenylphosphonium bromideand KO^(t)BU in THF gave the 17-methylidene compound 50. Azidation usingZnN₆.2py, PPh₃ and DIAD in toluene gave the 3α-azido compound 51.Lithium aluminum hydride reduction in THF gave the 3α-amino compound 52.Treatment with 80% acetic acid removed the acetonide protecting groupand formed the ammonium acetate salt 53. Alternatively compound 52 wastreated with hydrochloric acid in acetonitrile and water to give thehydrochloride salt 54.

Synthesis of Compound 50

A solution of KO^(t)Bu (2.0 g, 16.9 mmol), MePPh₃Br (6.0 g, 16.8 mmol)and 27 ml of THF was stirred at room temperature under argon. After 1hour the ketone 45 (2.00 g, 5.52 mmol) was added to the yellow solutionand the resulting solution was heated at reflux for 1 hour. The reactionwas quenched with 50 ml of brine, diluted with 100 ml of EtOAc,separated and washed with 25 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 1:1EtOAc/hexanes afforded 1.90 g (95%) of compound 50 as a white solid.

Synthesis of Compound 51

DIAD (0.85 ml, 4.10 mmol) was added dropwise over 15 minutes to a roomtemperature solution of the 3β-hydroxyl compound 50 (739 mg, 2.05 mmol),ZnN₆.2py (473 mg, 1.54 mmol), Ph₃P (1.075 g, 4.10 mmol) and toluene (20ml) under argon. After 4 hours the reaction mixture was loaded onto acolumn of silica gel packed in 10% ethyl acetate/hexanes and eluted with20% ethyl acetate/hexanes to afford 743 mg (94%) of compound 51 as awhite foam.

Synthesis of Compound 52

Lithium aluminum hydride (77 mg, 1.93 mmol) was added to an ice cooledsolution of the 3α-azide 51 in 5 ml of THF and 5 ml of diethyl etherunder argon. The reaction was allowed to warm to room temperature. After4 hours the solution was cooled in ice, diluted with 25 ml of diethylether and slowly quenched with 5 ml of saturated Na₂SO₄ solution. After10 minutes a white precipitate had formed and the solution was dilutedwith 50 ml of ethyl acetate, washed with 3×10 ml of brine, dried overMgSO₄, filtered and concentrated. The crude material was purified usinga column of silica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washingwith 5% MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, elutedwith 5% MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give 636 mg(92%) of compound 52 as a white solid.

Synthesis of Compound 53

A solution of the 3α-amine 52 (287 mg, 0.799 mmol) and 10 ml of 80%acetic acid was heated at 40° C. for 1 hour. The reaction mixture wasconcentrated to give a white foam. Acetone (10 ml) was added and thesolution was sonicated to dissolve the material and then evaporated.Another 10 ml portion of acetone was added, sonicated and evaporated togive 301 mg (99%) of compound 53 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.19;C₂₀H₃₄NO₂.

Synthesis of Compound 54

A solution of 4 M HCl in dioxane was added to a solution of the amine 52in 1 ml of acetonitrile and 50 μl of water. The resulting gummy solidwas diluted with 2 ml of acetonitrile and stirred vigorously until asolid formed. The solid was filtered and dried to give 50 mg (63%) ofcompound 54. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 320.19; C₂₀H₃₄NO₂.

Example 3 3-Amino-6,7-Dihydroxy-17-Methylidene Steroid (AlternativeSynthesis)

Intermediate 52 was also synthesized by the alternate route shown inScheme 3. Azidation of the alcohol 45 using ZnN₆.2py, PPh₃ and DIAD intoluene gave the 3α-azido compound 55. Hydrogenation of the azide, usingPd on carbon as catalyst gave the amine 56. Olefination of compound 56using methyltriphenylphosphonium bromide and KO^(t)Bu in THF gave the17-methylidene compound 52.

Synthesis of Compound 55

DIAD (2.4 ml, 11.6 mmol) was added dropwise over 20 minutes to a roomtemperature solution of the 3β-hydroxyl compound 45 (2.108 g, 5.81mmol), ZnN₆.2py (1.34 g, 4.36 mmol), Ph₃P (3.05 g, 11.6 mmol) andtoluene (58 ml) under argon. After being allowed to react overnight, thereaction mixture was loaded onto a column of silica gel and eluted with20% ethyl acetate/hexanes to afford 1.14 g (50%) of compound 55 as awhite foam.

Synthesis of Compound 56

A solution of the azide 55 (1.10 g, 2.84 mmol), 10% Pd on carbon (60 mg,0.057 mmol) and 28 ml of ethyl acetate was stirred at room temperatureovernight under hydrogen. The solution was filtered through celiteeluting with ethyl acetate. Purification by radial chromatographyeluting with 95:5:2 CH₂Cl₂:MeOH:Et₃N gave 892 mg (81%) of compound 56 asa white solid.

Synthesis of Compound 52

A solution of KO^(t)Bu (175 mg, 1.48 mmol), MePPh₃Br (528 mg, 1.48 mmol)and 3 ml of THF was stirred at room temperature under argon. After 1hour the ketone 56 (100 mg, 0.277 mmol) was added to the yellow solutionand the resulting solution was allowed to stir at room temperatureovernight. The reaction was quenched with 5 ml of water, diluted with 50ml of EtOAc, separated and washed with 10 ml of brine, dried over MgSO₄,filtered and concentrated. Purification by radial chromatography elutingwith 95:5:2 CH₂Cl₂:MeOH:Et₃N afforded 96 mg (97%) of compound 52 as awhite solid.

Example 4 3-Amino-6,7-Dihydroxy-17-Fluoroethylidene Steroid

Halogenated analogues related to compound 49 can be prepared usinghalogenated olefination reagents. Scheme 4 outlines the synthesis of the20-fluoro analogue 64. The hydroxyl in compound 45 was protected bytreatment with tert-butyldimethylsilyl chloride and imidazole indimethylformamide (DMF). Olefination of the ketone 57 using the anion oftriethyl 2-fluoro-2-phosphonoacetate gives a mixture of compound 58 andits geometric isomer. The compounds are separable using silica gelchromatography. Lithium aluminum hydride reduction of the ester in Et₂Ogave the allylic alcohol 59. Treatment with sulfur trioxide pyridinecomplex in THF followed by addition of lithium aluminum hydride affordsthe dehydroxylated compound 60. Tetrabutylammonium fluoride in THFremoves the protecting group from the 3-hydroxyl to give compound 61.Azidation using ZnN₆.2py, PPh₃ and DIAD in toluene gave the 3α-azidocompound 62. Lithium aluminum hydride reduction in THF gave the 3α-amine63. Treatment with HCl in THF and water deprotected the 6- and7-hydroxyls and formed the ammonium chloride salt 64.

Synthesis of Compound 57

A solution of the ketone 45 (4.73 g, 13.1 mmol), TBSCl (3.01 g, 19.6mmol), imidazole (2.67 g, 39.2 mmol) and DMF (52 ml) was stirred at roomtemperature overnight. The white slurry was diluted with 250 ml ofEtOAc, washed with 2×50 ml of water and 50 ml of brine, dried overMgSO₄, filtered and concentrated to give 5.97 g (96%) of compound 57 aswhite solid.

Synthesis of Compound 58

Lithium bis(trimethylsilyl)amide (10.0 ml of a 1.0 M solution in THF,10.0 mmol) was added to a room temperature solution of(EtO)₂P(O)CHFCO₂Et (2.65 g, 10.5 mmol) in THF (22 ml) under argon. After1 hour a solution of the ketone 57 (2.50 g, 5.25 mmol) in THF (20 ml)was added and the resulting solution was heated at reflux for 4.5 hoursand then stirred at room temperature overnight. The reaction wasquenched with 1.5 ml of saturated NaHCO₃ solution and then partiallyconcentrated to remove most of the THF. The residue was diluted with 200ml of EtOAc, washed with 3×20 ml of brine, dried over MgSO₄, filteredand concentrated. The crude material was purified by columnchromatography, eluting with 2.5% then 5% EtOAc/hexanes to give 1.50 g(50%) of compound 58 as a white solid.

Synthesis of Compound 59

Lithium aluminum hydride (106 mg, 2.66 mmol) was added to an ice cooledsolution of the ester 58 (1.50 g, 2.66 mmol) in Et₂O (13 ml) underargon. The solution was allowed to warm to room temperature. After 3hours the solution was cooled in ice and 20 ml of saturated Na₂SO₄solution was slowly added. After 10 minutes the solution was dilutedwith 150 ml of EtOAc, washed with water and brine, dried over MgSO₄,filtered and concentrated to give 1.44 g (quantitative) of compound 59as a white foam.

Synthesis of Compound 60

Sulfur trioxide pyridine complex (69.5 mg, 0.428 mmol) was added to anice cooled solution of the allylic alcohol 59 (149 mg, 0.285 mmol) inTHF (2.8 ml) under argon. After 6 hours lithium aluminum hydride (68 mg,1.71 mmol) was added and the solution was allowed to warm to roomtemperature overnight. The solution was cooled in ice and 5 ml ofsaturated Na₂SO₄ solution was slowly added. After 10 minutes thesolution was diluted with 75 ml of EtOAc, washed with water and brine,dried over MgSO₄, filtered and concentrated to give 109 mg (76%) ofcompound 60 as a white solid.

Synthesis of Compound 61

A solution of compound 60 (410 mg, 0.810 mmol), Bu₄NF (0.89 ml of a 1.0M solution in THF, 0.89 mmol) and THF (5 ml) was heated at reflux underargon. After 1.5 hours the solution was cooled to room temperature,diluted with 75 ml of EtOAc, washed with 20 ml of water and 2×20 ml ofbrine, dried over MgSO₄, filtered and concentrated. The residue wasfiltered through silica gel eluting with EtOAc and concentrated to give318 mg (100%) of compound 61 as a white solid.

Synthesis of Compound 62

DIAD (0.33 ml, 1.59 mmol) was added dropwise over 10 minutes to a roomtemperature solution of the 3β-alcohol 61 (312 mg, 0.796 mmol), ZnN₆.2py(183 mg, 0.597 mmol), Ph₃P (417 mg, 1.59 mmol) and toluene (8.0 ml)under argon. After 3 hours the reaction mixture was loaded onto a columnof silica gel packed in 10% EtOAc/hexanes and eluted with 20%EtOAc/hexanes to afford 322 mg (97%) of compound 62 as a crystallinesolid.

Is Synthesis of Compound 63

Lithium aluminum hydride (29 mg, 0.75 mmol) was added to an ice cooledsolution of the azide 62 (314 mg, 0.753 mmol) in 7.5 ml of Et₂O underargon. The reaction was allowed to warm to room temperature whilestirring overnight. The solution was cooled in ice and slowly quenchedwith 10 ml of saturated Na₂SO₄ solution. After 10 minutes a whiteprecipitate had formed and the solution was diluted with 75 ml of EtOAc,washed with 20 ml of water and 2×20 ml of brine, dried over MgSO₄,filtered and concentrated. The crude material was purified using acolumn of silica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washingwith 5% MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, elutedwith 5% MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give a whitesolid. ¹H NMR analysis indicated the material contained a trace of Et₃Ntherefore the material was taken up in 75 ml of CH₂Cl₂ and washed with2×25 ml of water, dried over MgSO₄, filtered and concentrated to give137 mg (47%) of compound 63 as a colorless film.

Synthesis of Compound 64

A solution of the 3α-amino compound 63 (137 mg, 0.35 mmol), 4 M HCl indioxane (105 μl, 0.42 mmol), THF (5.6 ml) and water (1.4 ml) was stirredat room temperature overnight. The solution was concentrated, theresidue was twice taken up in 3 ml of methanol and concentrated to give130 mg (96%) of compound 64 as an off-white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 352.14;C₂₁H₃₅FNO₂.

Example 5 3-Amino-6,7-Dihydroxy-17-Carbomethoxyethylidene Steroid

Olefination of compounds related to compound 57 can also be carried outto generate 21-carboalkoxy substituted analogues. Scheme 5 shows thesynthesis of the 21-carbomethoxy substituted example compound 69.Olefination of the ketone 57 using the anion of trimethyl2-phosphonoacetate gives a mixture of compound 65 and its geometricisomer. The compounds are separable using silica gel chromatography.Tetrabutylammonium fluoride in THF removes the protecting group from the3-hydroxyl to give compound 66. Azidation using ZnN₆.2py, PPh₃ and DIADin toluene gave the 3α-azido compound 67. Hydrogenation of the azide,using Pd on carbon as catalyst gave the 3α-amine 68. Treatment with 80%acetic acid deprotected the 6- and 7-hydroxyls and formed the ammoniumacetate salt 69.

Synthesis of Compound 65

Lithium bis(trimethylsilyl)amide (2.00 ml of a 1.0 M solution in THF,2.00 mmol) was added to a room temperature solution of(MeO)₂P(O)CH₂CO₂Me (390 mg, 2.10 mmol) in THF (22 ml) under argon. After3 hours a solution of the ketone 57 (509 mg, 1.07 mmol) in THF (2 ml)was added and the resulting solution was heated at reflux for 3 days.The reaction was quenched with 5 ml of water, diluted with 75 ml ofEtOAc, washed with 2×15 ml of brine, dried over MgSO₄, filtered andconcentrated. The crude material was purified by column chromatography,eluting with 5% EtOAc/hexanes to give 307 mg (54%) of compound 65 as acolorless film. Also isolated was 119 mg (21%) of the Z-isomer.

Synthesis of Compound 66

A solution of 65 (296 mg, 0.550 mmol), Bu₄NF (0.61 ml of a 1.0 Msolution in THF, 0.61 mmol) and THF (3 ml) was heated at reflux underargon. After 1 hour the solution was cooled to room temperature, dilutedwith 20 ml of EtOAc, washed with 10 ml of water and 2×10 ml of brine,dried over MgSO₄, filtered and concentrated to give 230 mg (100%) ofcompound 66 as a white solid.

Synthesis of Compound 67

DIAD (252 μl, 1.28 mmol) was added dropwise to a room temperaturesolution of the 3β-alcohol 66 (230 mg, 0.55 mmol), ZnN₆.2py (147 mg,0.48 mmol), Ph₃P (335 mg, 1.28 mmol) and toluene (6.4 ml) under argon.After 2 hours the reaction mixture was purified by radialchromatography, eluting with 15% EtOAc/hexanes to afford 203 mg (84%) ofcompound 67.

Synthesis of Compound 68

A solution of the azide 67 (203 mg, 0.45 mmol), 10% Pd on carbon (48 mg,0.045 mmol) and 4.5 ml of EtOAc was stirred at room temperature underhydrogen for 3 days. The solution was filtered through celite elutingwith EtOAc and MeOH to give 165 mg (88%) of compound 68.

Synthesis of Compound 69

A solution of the amine 68 (165 mg, 0.40 mmol) and 2 ml of 80% aceticacid was heated at 40° C. for 1 hour. The reaction mixture was dilutedwith 10 ml of toluene then concentrated to remove residual acetic acid.Trituration of the residue in 10 ml of cyclohexane, followed byfiltration and drying gave 117 mg (67%) of compound 69 as a white solid.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 378.17; C₂₂H₃₆NO₄.

Example 6 3α-Amino-6α,7β-Dihydroxyandrostan-17-One Acetic Acid Salt

Analogous methodology can be used to obtain compounds with differentfunctionalities at C17. For example a 17-ketone substituted compound isobtained by treatment of compound 56 with 80% acetic acid to give3α-amino-6α,7β-dihydroxyandrostan-17-one acetic acid salt (70) (seeTable 2).

Synthesis of Compound 70

A solution of the ketone 56 (67 mg, 0.16 mmol) and 1 ml of 80% aceticacid was heated at 40° C. for 1 hour. The reaction mixture was dilutedwith 10 ml of toluene then concentrated to give 63 mg (100%) of compound70. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 waterand MeCN) 322.18; C₁₉H₃₂NO₃.

Example 7 3-Amino-6,7-Dihydroxy-17-Hydroxy Steroid

17-Hydroxyl substituted analogues can be prepared from ketones relatedto compound 56 as shown in Scheme 6. The carbonyl in compound 56 wasreduced with NaBH₄ in methanol to give exclusively the 17β-hydroxylisomer 71. Treatment with 80% acetic acid removed the acetonideprotecting group and formed the ammonium acetate salt 72.

Synthesis of Compound 71

An ice cooled solution of the ketone 56 (100 mg, 0.28 mmol), NaBH₄ (16mg, 0.41 mmol) and 1.4 ml of MeOH was allowed to react for 2.5 hours.The reaction was quenched by the addition of 1 ml of water andconcentrated to remove most of the MeOH. The residue was diluted with 40ml of CH₂Cl₂ and washed with 2×10 ml of brine, dried over MgSO₄,filtered and concentrated to give 90 mg (90%) of compound 71.

Synthesis of Compound 72

A solution of the amine 71 (90 mg, 0.25 mmol) and 2 ml of 80% aceticacid was heated at 40° C. for 2 hours. The reaction mixture was twicediluted with 10 ml of toluene and concentrated to remove residual aceticacid. The residue was dissolved in 1 ml of MeOH and 5 ml of hexanes,concentrated and dried to give 86 mg (90%) of compound 72 as a whitesolid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1water and MeCN) 324.19; C₁₉H₃₄NO₃.

Example 8 Salts of 3α-Amino-6,7-Dihydroxy-17-Methylidene Steroid

The 6- and 7-hydroxyls can be protected using a variety of protectinggroups. Suitable protecting groups are listed in Greene and Wuts,“Protective Groups in Organic Synthesis”, John Wiley & Sons, New York,N.Y. (1999). Scheme 7 shows examples of analogues that have beensynthesized with the 6- and 7-hydroxyls protected as methyl ethers. Thestarting material compound 73 for the synthesis is described in U.S.Pat. No. 6,046,185. Generation of the dianion of compound 73 using NaHin dimethylformamide followed by alkylation with methyl iodide gavecompound 74. Treatment with 80% acetic acid removed both the cyclicketal and tert-butyldimethylsilyl ether protecting groups. Olefinationof compound 75 using methyltriphenylphosphonium bromide and KO^(t)Bu inTHF gave the 17-methylidene compound 76. Azidation using ZnN₆.2py, PPh₃and DIAD in toluene gave the 3α-azido compound 77. Lithium aluminumhydride reduction in THF gave the 3α-amine 78. Treatment with HCl inEt₂O and MeOH formed the ammonium chloride salt 79. Treatment ofcompound 78 with acetic acid formed the ammonium acetate salt 80.

Synthesis of Compound 74

Sodium hydride (0.50 g, 12.4 mmol) was added to a room temperaturesolution of the diol 73 (1.49 g, 3.10 mmol) in 15 ml of DMF undernitrogen. After 2 hours the solution was cooled in ice and MeI (1.93 ml,30.9 mmol) was added dropwise over 30 seconds. The reaction was allowedto warm to room temperature while stirring overnight. The reactionmixture was diluted with 100 ml of Et₂O, washed with 10 ml of water and2×10 ml of brine, dried over MgSO₄, filtered and concentrated to give1.73 g of crude compound 74 as a pale yellow oil.

Synthesis of Compound 75

A solution of crude compound 74 (1.73 g, 3.10 mmol) and 15 ml of 80%acetic acid was stirred at room temperature for 4 hours. The solutionwas concentrated, the residue taken up in 50 ml of EtOAc, washed with2×20 ml of saturated NaHCO₃ solution and 2×10 ml of brine, dried overMgSO₄, filtered and concentrated to give 1.18 g of crude compound 75 asa white foam.

Synthesis of Compound 76

A solution of KO^(t)Bu (1.09 g, 9.20 mmol), MePPh₃Br (3.30 g, 9.20 mmol)and 15 ml of THF was stirred at room temperature under nitrogen. After 2hours crude ketone 75 (1.17, mg, 3.08 mmol) was added to the yellowsolution and the resulting solution was allowed to stir at roomtemperature overnight. The reaction was quenched with 2 ml of water,diluted with 100 ml of EtOAc, washed with 3×10 ml of brine, dried overMgSO₄, filtered and concentrated. Column chromatography eluting with 80%EtOAc/hexanes afforded 890 mg of impure compound 76 as a white solid.

Synthesis of Compound 77

DIAD (1.05 ml, 5.08 mmol) was added dropwise over 10 minutes to a roomtemperature solution of the 3β-alcohol 76 (885 mg, 2.54 mmol), ZnN₆.2py(585 mg, 1.90 mmol), Ph₃P (1.33 g, 5.08 mmol) and toluene (25 ml) underargon. After 11 hours the reaction mixture was purified by columnchromatography eluting with 15% EtOAc/hexanes to afford 594 mg (63%) ofcompound 77 as a crystalline solid.

Synthesis of Compound 78

Lithium aluminum hydride (0.79 ml of a 1 M solution in Et₂O, 0.79 mmol)was added to an ice cooled solution the azide 77 (588 mg, 1.57 mmol) in15.7 ml of Et₂O under argon. After 10 minutes the reaction was allowedto warm to room temperature while stirring overnight. After 1 hour thereaction mixture was cooled in ice and slowly quenched with 10 ml ofsaturated Na₂SO₄ solution. After 10 minutes a white precipitate hadformed and the liquid was decanted off. The residue was washed with 2×25ml of EtOAc and the washings were combined with the previously decantedether solution. The solution was washed with 3×10 ml of brine, driedover MgSO₄, filtered and concentrated. Purification by columnchromatography eluting with 95:5:2 CH₂Cl₂:MeOH:Et₃N gave 434 mg (79%) ofcompound 78 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂.

Synthesis of Compound 79

Hydrogen chloride (0.26 ml of a 1.0 M solution in Et₂O, 0.26 mmol) wasadded to a solution of the amine 78 (60 mg, 0.17 mmol) in 2 ml of Et₂O.The resulting gel-like material was dissolved in 5 ml of methanol andconcentrated. The residue was dissolved in 1 ml of methanol, dilutedwith 5 ml of cyclohexane and concentrated to give 66 mg (100%) ofcompound 79 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂.

Synthesis of Compound 80

A solution of the amine 78 (61 mg, 0.17 mmol) and 1 ml of acetic acidwas allowed to stand at room temperature for 30 minutes. The solutionwas diluted with 5 ml of toluene and concentrated. The residue was takenup in 5 ml of hexanes, concentrated and the residue was dried for 2hours using an Abderhalden drying apparatus with refluxing acetone togive 71 mg (100%) of compound 80 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 348.20;C₂₂H₃₈NO₂.

Example 9 Salts of 3β-Amino-6,7-Dihydroxy-17-Methylidene Steroid

The stereochemistry at C3 can be inverted to give 3β-ammonium saltderivatives of any number of compounds related to compound 49. Thestereochemistry at C3 can be inverted in 3 synthetic steps as shown inScheme 8 for the synthesis of compounds 28 and 83. The 3β-hydroxylcompound 46 is converted to the 3β-mesylate 81 using methanesulfonylchloride and pyridine. Heating compound 81 and cesium acetate in 100° C.DMF gives the 3α-acetate compound 82. The inversion sequence iscompleted by methanolysis of the acetate in compound 82 using sodiummethoxide to give the 3α-hydroxyl compound 25. Treatment of compound 25with ZnN₆.2py, triphenylphosphine and DIAD in toluene produced the3β-azido compound 26. Lithium aluminum hydride reduction of the azide inEt₂O provided the 3β-amino compound 27. Treatment with HCl in THF andwater removes the acetonide group and forms the ammonium chloride salt28. Similarly, treatment of compound 27 with 80% acetic acid removes theacetonide group and forms the ammonium acetate salt 83. Using themethods outlined in Scheme 8, compound 50 is converted into compound 89and compound 61 is converted into compound 95 (see Table 1). Compounds26, 27, 87, 88, 89, 93, 94 and 95 are examples of compounds of theinvention having 3β stereochemistry.

Synthesis of Compound 81

Methanesulfonyl chloride (1.2 ml, 16 mmol) was added to an ice cooledsolution of the 3β-hydroxyl compound 46 (3.0 g, 8.0 mmol) in pyridine(20 ml) under argon. After 4 hours the solution was cooled in ice and 20ml of saturated NaHCO₃ solution was added. After 15 minutes the solutionwas diluted with 150 ml of EtOAc and washed with 3×25 ml of brine, driedover MgSO₄, filtered and concentrated to give 3.6 g (100%) of compound81 as an off-white foam.

Synthesis of Compound 82

A solution of the mesylate 81 (3.6 g, 8.0 mmol), cesium acetate (4.6 g,24 mmol) and 40 ml of DMF was heated at 100° C. for 24 hours. Thesolution was diluted with 100 ml of water, extracted with 2×100 ml ofEt₂O, washed with 2×50 ml of brine, dried over MgSO₄, filtered andconcentrated to give approximately 3 g of crude compound 82.

Synthesis of Compound 25

A solution of Na (398 mg, 17.3 mmol) in MeOH (21.5 ml) was added to the3α-acetate 82 (1.8 g, 4.3 mmol) in THF (10 ml). After 2 hours, 20 ml ofwater was added and the resulting solution was diluted with 100 ml ofEtOAc, washed consecutively with saturated NaHCO₃ solution, water andbrine, dried over MgSO₄, filtered and concentrated to give 1.58 g (98%)of crude compound 25 as a yellow foam.

Synthesis of Compound 26

DIAD (1.70 ml, 8.24 mmol) was added dropwise over 10 minutes to a roomtemperature solution of the 3α-alcohol 25 (1.54 g, 4.12 mmol), ZnN₆.2py(0.94 g, 3.09 mmol), Ph₃P (2.16 g, 8.24 mmol) and toluene (44 ml) underargon. After stirring overnight, the reaction mixture was loaded onto acolumn of silica gel packed in 10% EtOAc/hexanes and eluted with 10%EtOAc/hexanes to afford 0.89 g (61%) of compound 26 as a white solid.

Synthesis of Compound 27

Lithium aluminum hydride (146 mg, 3.66 mmol) was added to an ice cooledsolution the azide 26 (1.46 g, 3.66 mmol) in 18.3 ml of Et₂O underargon. The reaction was allowed to warm to room temperature. After 1.5hours the solution was cooled in ice, diluted with 25 ml of Et₂O andslowly quenched with 20 ml of saturated Na₂SO₄ solution. After 10minutes a white precipitate had formed and the solution was diluted with50 ml of EtOAc, washed with 3×10 ml of brine, dried over MgSO₄, filteredand concentrated to give 1.31 g (96%) of compound 27 as a white foam.

Synthesis of Compound 28

A solution of the 3β-amino compound 27 (227 mg, 0.609 μmmol), 4 M HCl indioxane (183 μl, 0.73 mmol), THF (9.7 ml) and water (2.4 ml) was stirredat room temperature overnight. Evaporated the THF and water, took up theresidue in 5 ml of methanol and concentrated, triturated with 5 ml ofacetone, concentrated to give 224 mg (100%) of compound 28 as a whitesolid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1water and MeCN) 334.10; C₂₁H₃₆NO₂.

Synthesis of Compound 83

A solution of the amine 27 (412 mg, 1.10 mmol) and 5 ml of 80% aceticacid was stirred at room temperature for 4 hours. The reaction mixturewas diluted with 5 ml of toluene then concentrated. The residue wastwice more taken up in 5 ml portions of toluene and concentrated toremove residual acetic acid. The residue was twice triturated in 10 mlof CH₂Cl₂ and concentrated to give 430 mg (99%) of compound 83 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 334.19; C₂₁H₃₆NO₂.

Synthesis of Compound 84

Methanesulfonyl chloride (0.33 ml, 4.2 mmol) was added to an ice cooledsolution of the 3β-hydroxyl compound 50 (754 mg, 2.09 mmol) in pyridine(5.3 ml) under argon. After 4 hours the solution was cooled in ice and 5ml of saturated NaHCO₃ solution was added. After 15 minutes the solutionwas diluted with 60 ml of ethyl acetate and washed with 3 times withbrine, dried over MgSO₄, filtered and concentrated to give 860 mg (94%)of compound 84 as an off-white solid

Synthesis of Compound 85

A solution of the mesylate 84 (860 mg, 1.96 mmol), cesium acetate (1.13g, 5.88 mmol) and 10 ml of DMF was heated at 95° C. for 32 hours. Thesolution was diluted with 50 ml of water, extracted with 2×100 ml ofEt₂O, washed with 2×30 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 5% and8% EtOAc/Hexanes afforded 558 mg (71%) of compound 85 as white solid.

Synthesis of Compound 86

A solution of Na (128 mg, 5.56 mmol) in MeOH (7 ml) was added to the3α-acetate 85 (558 mg, 1.38 mmol). After 2 hours 5 ml of saturatedNaHCO₃ solution was added and the resulting solution was diluted with100 ml of EtOAc. The solution washed with 2×20 ml of water and 2×20 mlof brine, dried over MgSO₄, filtered and concentrated to give 491 mg(99%) of compound 86 as a white solid.

Synthesis of Compound 87

DIAD (0.57 ml, 2.74 mmol) was added dropwise over 15 minutes to a roomtemperature solution of the 3α-hydroxy compound 86 (493 mg, 1.37 mmol),ZnN₆.2py (315 mg, 1.03 mmol), Ph₃P (718 mg, 2.74 mmol) and toluene (13.7ml) under argon. After 3.5 hours the reaction mixture was loaded onto acolumn of silica gel packed in 10% ethyl acetate/hexanes and eluted with10% ethyl acetate/hexanes to afford 390 mg (74%) of compound 87 as aviscous oil.

Synthesis of Compound 88

Lithium aluminum hydride (40 mg, 1.01 mmol) was added to an ice cooledsolution of the azido compound 87 (390 mg, 1.01 mmol) in 5 ml of diethylether under argon. The reaction was allowed to warm to room temperature.After 2 hours the solution was cooled in ice, diluted with 25 ml ofdiethyl ether and slowly quenched with 2 ml of saturated Na₂SO₄solution. After 10 minutes a white precipitate had formed and thesolution was diluted with 40 ml of ethyl acetate, washed with 3×15 ml ofbrine, dried over MgSO₄, filtered and concentrated. The crude materialwas purified using a column of silica gel prepared by packing in 1%Et₃N/CH₂Cl₂ and washing with 5% MeOH/CH₂Cl₂. The crude material wasloaded in CH₂Cl₂, eluted with 5% MeOH/CH₂Cl₂ and then 95:5:2CH₂Cl₂:MeOH:Et₃N to give 277 mg (76%) of compound 88 as a white solid.

Synthesis of Compound 89

A solution of the amino compound 88 (270 mg, 0.752 mmol) and 10 ml of80% acetic acid was heated at 40° C. for 1 hour. The reaction mixturewas concentrated to give a white foam. Acetone (10 ml) was added andsonicated to dissolve the material and then evaporated. Another 10 mlportion of acetone was added, sonicated and evaporated to give 285 mg(100%) of compound 89 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.26; C₂₀H₃₄NO₂.

Synthesis of Compound 90

Methanesulfonyl chloride (0.20 ml, 2.56 mmol) was added to an ice cooledsolution of the 3β-hydroxyl compound 61 (501 mg, 1.28 mmol) in pyridine(3.2 ml) under argon. After 4 hours the solution was cooled in ice and 5ml of saturated NaHCO₃ solution was added. After 15 minutes the solutionwas diluted with 50 ml of EtOAc and washed 3 times with brine, driedover MgSO₄, filtered and concentrated to give 590 mg (98%) of compound90 as a white foam.

Synthesis of Compound 91

A solution of the mesylate 90 (590 mg, 1.25 mmol), cesium acetate (722mg, 3.76 mmol) and 6.2 ml of DMF was heated at 100° C. for 24 hours. Thesolution was diluted with 50 ml of water, extracted with 2×50 ml ofEt₂O, washed with 2×30 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification by column chromatography eluting with 8%EtOAc/hexanes afforded 297 mg (55%) of compound 91 as a white solid.

Synthesis of Compound 92

A solution of Na (63 mg, 2.7 mmol) in MeOH (3.4 ml) was added to the3α-acetate 91 (297 mg, 0.684 mmol) in THF (1 ml). The solution wasstirred overnight, 5 ml of water and 80 ml of EtOAc were added andwashed twice with water and twice with brine, dried over MgSO₄, filteredand concentrated to give 251 mg (94%) of compound 92 as a white solid.

Synthesis of Compound 93

DIAD (0.26 ml, 1.24 mmol) was added dropwise over 10 minutes to a roomtemperature solution of the 3α-alcohol 92 (243 mg, 0.620 mmol), ZnN₆.2py(143 mg, 0.465 mmol), Ph₃P (325 mg, 1.24 mmol) and toluene (6.2 ml)under argon. After 4 hours the reaction mixture was loaded onto a columnof silica gel packed in 10% EtOAc/hexanes and eluted with 20%EtOAc/hexanes to afford 209 mg of impure compound 93 as a yellow oil.

Synthesis of Compound 94

Lithium aluminum hydride (20 mg, 0.50 mmol) was added to an ice cooledsolution the impure azide 93 (209 mg, 0.50 mmol) in 5 ml of Et₂O underargon. The reaction was allowed to warm to room temperature. After 4hours the solution was cooled in ice, diluted with 25 ml of Et₂O andslowly quenched with 2 ml of saturated Na₂SO₄ solution. After 10 minutesa white precipitate had formed and the solution was diluted with 50 mlof EtOAc, washed with 3×10 ml of brine, dried over MgSO₄, filtered andconcentrated. The crude material was chromatographed using a column ofsilica gel prepared by packing in 1% Et₃N/CH₂Cl₂ and washing with 5%MeOH/CH₂Cl₂. The crude material was loaded in CH₂Cl₂, eluted with 5%MeOH/CH₂Cl₂ and then 95:5:2 CH₂Cl₂:MeOH:Et₃N to give 97 mg of impurecompound 94 as a white solid.

Synthesis of Compound 95

A solution of the impure 3β-amino compound 94 (97 mg, 0.25 mmol), 4 MHCl in dioxane (74 μl, 0.30 mmol), THF (4 ml) and water (1 ml) wasstirred at room temperature. After 4 hours the solution wasconcentrated, the residue was taken up in 5 ml of methanol andconcentrated. The residue was twice triturated with 5 ml of acetone andconcentrated. The white solid was dissolved in approximately 0.5 ml ofwater and acetone (5 ml) was slowly added until crystals appeared. Thecrystals were filtered, rinsed with acetone and dried to give 66 mg ofcompound 95 as colorless fine needles. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 352.09;C₂₁H₃₅FNO₂.

Example 10 3-Amino-6,7-Dihydroxy-17-Alkyl Steroid

Any compounds having the 17(20)-alkenyl functionality can have thedouble bond hydrogenated using H₂ in the presence of a catalyst such as10% Pd on carbon. For example compound 96 has been prepared fromcompound 28 as shown in Scheme 9. Similarly, compound 97 was preparedfrom compound 49 using the same methodology as shown in Scheme 9 (seeTable 2).

Synthesis of Compound 96

A solution of the olefin 28 (52 mg, 0.14 mmol), 10% Pd on carbon (15 mg,0.014 mmol) and methanol (3 ml) was stirred at room temperatureovernight under hydrogen. The solution was filtered through celiteeluting with 50 ml of methanol and concentrated to give 50 mg (96%) ofcompound 96 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 336.24; C₂₁H₃₈NO₂.

Synthesis of Compound 97

A solution of the olefin 49 (844 mg, 2.28 mmol), 10% Pd on carbon (243mg, 0.228 mmol) and methanol (11 ml) was stirred at room temperatureovernight under hydrogen. The solution was filtered through celiteeluting with 50 ml of methanol and concentrated. The residue wastriturated in 10 ml of acetone, filtered and dried to give 801 mg (94%)of compound 97 as a white solid. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 336.21; C₂₁H₃₈NO₂.

Example 11 3-Secondary Amino-6,7-Dihydroxy-17-Methylidene Steroid

Any amine related to compound 52 can be coupled to an aldehyde or ketoneto prepare secondary or tertiary amines. Reaction of compound 52 with asolution of 4-isopropylbenzaldehyde and titanium isopropoxide in THFfollowed by reduction with sodium borohydride gives compound 99.Treatment with 80% acetic acid removes the acetonide group and forms theammonium acetate salt 100. Example compounds 101-107 were synthesizedusing the methods outlined in Scheme 10 (see Table 6).

Synthesis of Compound 99

Titanium(IV) isopropoxide (120 μl, 0.42 mmol) was added to a roomtemperature solution of the amine 52 (100 mg, 0.28 mmol),4-isopropylbenzaldehyde (46 μl, 0.31 mmol) and 1.4 ml of THF undernitrogen. After 12 hours a solution of NaBH₄ (29 mg, 0.78 mmol) in 1 mlof EtOH was added and the reaction was continued for another 8 hours.The reaction was quenched by the addition of 3 ml of brine, diluted with30 ml of EtOAc, separated, washed with 10 ml of brine, dried over MgSO₄,filtered and concentrated. Purification using radial chromatographyafforded 50 mg (36%) of compound 99.

Synthesis of Compound 100

A solution of the amine 99 (50 mg, 0.10 mmol) and 1 ml of 80% aceticacid was heated at 40° C. for 3 hours. The reaction mixture was twicetaken up in 5 ml portions of toluene and concentrated and then once eachwith acetone and hexanes to give 25 mg (51%) of compound 100. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)452.27; C₃₀H₄₆NO₂.

Synthesis of Compound 101

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with 2-fluorobenzaldehyde (32μl, 0.32 mmol) to give 43 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 49 mg (37%) of compound 101 asa white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 428.22; C₂₇H₃₉FNO₂.

Synthesis of Compound 102

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with3-(trifluoromethyl)benzaldehyde (41 μl, 0.31 mmol) to give 61 mg ofamine intermediate. The amine intermediate was treated with 1 ml of 80%acetic acid at 40° C. for 3 hours. The reaction mixture was diluted with5 ml of toluene and concentrated. The residue was dissolved in 1 ml ofacetone, diluted with 5 ml of hexanes and concentrated to give 64 mg(45%) of compound 102 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 478.18;C₂₈H₃₉F₃NO₂.

Synthesis of Compound 103

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with o-anisaldehyde (42 mg,0.31 mmol) to give 30 mg of amine intermediate. The amine intermediatewas treated with 1 ml of 80% acetic acid at 40° C. for 3 hours. Thereaction mixture was diluted with 5 ml of toluene and concentrated. Theresidue was dissolved in 1 ml of acetone, diluted with 5 ml of hexanesand concentrated to give 18 mg (14%) of compound 103. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 440.23;C₂₈H₄₂NO₃.

Synthesis of Compound 104

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with4-(trifluoromethoxy)benzaldehyde (44 μl, 0.31 mmol) to give 86 mg ofamine intermediate. The amine intermediate was treated with 1.5 ml of80% acetic acid at 40° C. for 3 hours. The reaction mixture was dilutedwith 5 ml of toluene and concentrated. The residue was dissolved in 1 mlof acetone, diluted with 5 ml of hexanes and concentrated to give 84 mg(57%) of compound 104 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 494.15;C₂₈H₃₉F₃NO₃.

Synthesis of Compound 105

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with 3-phenoxybenzaldehyde (60mg, 0.32 mmol) to give 73 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 87 mg (58%) of compound 105 asa white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 502.20; C₃₃H₄₄NO₃.

Synthesis of Compound 106

Using the procedure described for the synthesis of compound 99, theamine 52 (100 mg, 0.28 mmol) was reacted with 3-nitrobenzaldehyde (46mg, 0.31 mmol) to give 18 mg of amine intermediate. The amineintermediate was treated with 1 ml of 80% acetic acid at 40° C. for 3hours. The reaction mixture was diluted with 5 ml of toluene andconcentrated. The residue was dissolved in 1 ml of acetone, diluted with5 ml of hexanes and concentrated to give 18 mg (14%) of compound 106 asan off-white solid. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 455.20; C₂₇H₃₉N₂O₄.

Synthesis of Compound 107

Using the procedure described for the synthesis of compound 99, theamine 52 (200 mg, 0.55 mmol) was reacted with 3-pyridylcarboxaldehyde(82 μl, 0.61 mmol) to give 100 mg of amine intermediate. A suspension ofthe amine intermediate, 4 M HCl in dioxane (65 μl, 0.26 mmol), 110 μl ofwater and 2.2 ml of acetonitrile was stirred at room temperature for 1hour. The solution was filtered and the solid was dried to afford 77 mg(30%) of compound 107 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 411.21;C₂₆H₃₉N₂O₂.

Example 12 3-Cycloamino-6,7-Dihydroxy-17-Ethylidene Steroid

Any ketone related to compound 108 may be coupled to an amine using themethodology shown in Scheme 11. The starting material compound 108 forthe synthesis is described in U.S. Pat. No. 6,046,185. Reaction ofcompound 108 with piperidine and sodium cyanoborohydride in methanolgave compound 109 as a mixture of isomers at C3. Treatment with 80%acetic acid removed the acetonide protecting group and formed theammonium acetate salt 110. Example compound 111 was synthesized usingthe methods outlined in Scheme 11, except hydrochloric acid is used inplace of acetic acid (see Table 5). A 3-cycloamino group is a groupattached to the 3-position, where the carbon at the 3-position isattached directly to a nitrogen, and this nitrogen is part of aheterocyclic ring.

Synthesis of Compound 109

A solution of the ketone 108 (200 mg, 0.54 mmol), piperidine (266 μl,2.68 mmol), 100 mg of 3 Å molecular sieves, NaBH₃CN (24 mg, 0.38 mmol)and 5.4 ml of MeOH was stirred at room temperature for 24 hours. Thereaction mixture was diluted with 20 ml of water and extracted with 2×20ml of CH₂Cl₂. The combined extractions were washed with 10 ml of brine,dried over MgSO₄, filtered and concentrated. The crude material waspurified using radial chromatography eluting with 20% MeOH/CH₂Cl₂ toafford 112 mg (47%) of compound 109 as a white solid.

Synthesis of Compound 110

A solution of the amines 109 (102 mg, 0.23 mmol) and 5 ml of 80% aceticacid was heated at 40° C. for 1 hour. The solution was concentrated, theresidue was taken up in 2 ml of MeOH, diluted with 15 ml of toluene andconcentrated. The residue was triturated in 5 ml of acetone, filteredand dried to give 44 mg (42%) of compound 110 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)402.31; C₂₆H₄₄NO₂.

Synthesis of Compound 111

Using the procedure described for the synthesis of compound 109, theketone 108 (200 mg, 0.54 mmol) was reacted with morpholine (234 μl, 2.68mmol) to give 56 mg of the amine intermediate. The amine intermediatewas treated with 5 ml of 80% acetic acid at 40° C. for 1 hour. Thesolution was concentrated, dissolved in 5 ml of MeOH and concentrated.¹H and ¹³C NMR analyses indicated the acetonide protecting group hadbeen removed but little or none of the salt had formed. The material wastreated with 4 M HCl in dioxane (32 μl, 0.13 mmol) and 2 ml of acetonegiving a white precipitate. The suspension was diluted with 2 ml ofacetone, filtered and dried to give 48 mg (20%) of compound 111 as awhite solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 404.20; C₂₅H₄₂NO₃.

Example 13 3-Oxo to 3-Secondary Amino Conversion in Steroid

Any ketone related to compound 108 can be coupled to an amine using themethodology shown in Scheme 12. Methylamine is added to a solution ofcompound 108 and titanium isopropoxide in THF, followed by reductionwith sodium borohydride. The solution is filtered and eluted throughMP-TsOH resin to give compound 112, as a mixture of isomers at C3.Treatment with HCl in acetonitrile and water formed the ammoniumchloride salt 113. Example compounds 114-129 were synthesized using themethods outlined in Scheme 12, except that acetic acid was used in placeof hydrochloric acid for the examples in which ammonium acetate saltswere formed (see Table 5).

Synthesis of Compound 112

Titanium(IV) isopropoxide (270 μl, 0.92 mmol) was added to a roomtemperature solution of the ketone 108 (250 mg, 0.67 mmol), methylaminehydrochloride (41 mg, 0.61 mmol) and 1.5 ml of THF under nitrogen. After12 hours a solution of NaBH₄ (65 mg, 1.7 mmol) in 2.3 ml of EtOH wasadded and the reaction was continued for another 10 hours. The reactionwas quenched by the addition of 0.5 ml of water and filtered to remove awhite precipitate. The solution was loaded onto a column of 600 mg ofMP-TsOH resin and eluted with 3 ml of MeOH then 4 ml of 2 M NH₃ in MeOH.The NH₃/MeOH fraction was concentrated to give 76 mg (32%) of compound112.

Synthesis of Compound 113

A suspension of compound 112 (76 mg, 0.21 mmol), 4 M HCl in dioxane (75μl, 0.30 mmol), 50 μl of water and 1 ml of acetonitrile was stirred atroom temperature for 1 hour. The solution was filtered and the solid wasdried to afford 38 mg (13%) of compound 113 as a grey solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)348.19; C₂₂H₃₈NO₂.

Synthesis of Compound 114

Using the procedures described for the synthesis of compound 112, theketone 108 (200 mg, 0.53 mmol) was reacted with propylaminehydrochloride (47 mg, 0.49 mmol) to give 72 mg of amine intermediate.The amine intermediate was treated with 75 μl of the HCl solution togive 34 mg (17%) of compound 114. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 376.22; C₂₄H₄₂NO₂.

Synthesis of Compound 115

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with amylamine (70 μl, 0.61mmol) to give 82 mg of amine intermediate. The amine intermediate wastreated with 75 μl of the HCl solution to give 75 mg (28%) of compound115 as a white solid. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 404.28; C₂₆H₄₆NO₂.

Synthesis of Compound 116

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with cyclopentylamine (60 μl,0.61 mmol) to give 99 mg of amine intermediate. The amine intermediatewas treated with 200 μL of acetic acid for 1 hour and was twice taken upand concentrated from 1 ml portions of toluene. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to give 98 mg(35%) of compound 116. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 402.27; C₂₆H₄₄NO₂.

Synthesis of Compound 117

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with cyclohexylamine (70 μl,0.61 mmol) to give 120 mg of amine intermediate. The amine intermediatewas treated with 0.5 ml of acetic acid for 1 hour and was twice taken upand concentrated from 1 ml portions of toluene. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to give 101 mg(32%) of compound 117. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 416.25; C₂₇H₄₆NO₂.

Synthesis of Compound 118

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with pyrrolidine (51 μl, 0.61mmol) to give 70 mg of amine intermediate. The amine intermediate wastreated with 50 μl of acetic acid for 1 hour and 1 ml of cyclohexane wasadded to give a solid, which was filtered and dried to afford 65 mg(22%) of compound 118. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 388.28; C₂₅H₄₂NO₂.

Synthesis of Compound 119

Using the procedures described for the synthesis of compound 112, theketone 108 (200 mg, 0.53 mmol) was reacted with N-propylethylenediamine(60 μl, 0.49 mmol) to give 99 mg of amine intermediate. The amineintermediate was treated with 75 μl of the HCl solution to give 47 mg(20%) of compound 119. LC/MS (direct infusion, electrospray +ve, 10 mMNH₄OAc in 4:1 water and MeCN) 419.32; C₂₆H₄₇N₂O₂.

Synthesis of Compound 120

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted withN,N-dimethylethylenediamine (65 μl, 0.61 mmol) to give 93 mg of amineintermediate. The amine intermediate was treated with 75 μl of the HClsolution to give 77 mg (29%) of compound 120 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)405.28; C₂₅H₄₅N₂O₂.

Synthesis of Compound 121

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with piperazine (52 mg, 0.61mmol) to give 33 mg of amine intermediate. The amine intermediate wastreated with 200 μl of acetic acid for 1 hour and was twice taken up andconcentrated from 1 ml portions of toluene. The residue was trituratedin 1 ml of cyclohexane, filtered and dried to give 39 mg (14%) ofcompound 121. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 403.23; C₂₅H₄₃N₂O₂.

Synthesis of Compound 122

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with ethanolamine (33 μl,0.61 mmol) to give 136 mg of amine intermediate. The amine intermediatewas treated with 75 μl of the HCl solution to give 124 mg (50%) ofcompound 122 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 378.19; C₂₃H₄₀NO₃.

Synthesis of Compound 123

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with 5-amino-1-pentanol (63mg, 0.61 mmol) to give 129 mg of amine intermediate. The amineintermediate was treated with 75 μl of the HCl solution to give 65 mg(24%) of compound 123 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 420.25; C₂₆H₄₆NO₃.

Synthesis of Compound 124

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with2-(2-aminoethylamino)ethanol (62 μl, 0.61 mmol) to give 90 mg of amineintermediate. The amine intermediate was treated with 75 μl of the HClsolution to give 79 mg (28%) of compound 124 as a white solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)421.24; C₂₅H₄₅N₂03.

Synthesis of Compound 125

Using the procedures described for the synthesis of compound 112, theketone 108 (200 mg, 0.53 mmol) was reacted with m-toluidine (52 μl, 0.49mmol) to give 95 mg of amine intermediate. The amine intermediate wastreated with 75 μl of the HCl solution to give 43 mg (19%) of compound125. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 waterand MeCN) 424.23; C₂₈H₄₂NO₂.

Synthesis of Compound 126

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with 4-aminophenol (67 mg,0.61 mmol) to give 138 mg of amine intermediate. The amine intermediatewas treated with 75 μl of the HCl solution to give 41 mg (14%) ofcompound 126. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 426.18; C₂₇H₄₀NO₃.

Synthesis of Compound 127

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with sulfanilamide (105 mg,0.61 mmol) to give, after purification using radial chromatography, 24mg of amine intermediate. The amine intermediate was treated with 75 μlof the HCl solution to give 23 mg (7%) of compound 127. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 489.17;C₂₇H₄₁N₂O₄S.

Synthesis of Compound 128

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with 3-aminomethylpyridine(62 μl, 0.61 mmol) to give 108 mg of amine intermediate. The amineintermediate was reacted with 1 ml of 80% acetic acid at 40° C. for 1hour. The reaction mixture was concentrated and was twice taken up andconcentrated from 1 ml portions of toluene. The residue was trituratedin 1 ml of cyclohexane, filtered and dried to give 117 mg (41%) ofcompound 128. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in4:1 water and MeCN) 425.24; C₂₇H₄₁N₂O₂.

Synthesis of Compound 129

Using the procedures described for the synthesis of compound 112, theketone 108 (250 mg, 0.67 mmol) was reacted with histamine (68 mg, 0.61mmol) to give 120 mg of amine intermediate. The amine intermediate wasreacted with 1 ml of 80% acetic acid at 40° C. for 1 hour. The reactionmixture was concentrated and was twice taken up and concentrated from 1ml portions of toluene. The residue was triturated in 1 ml ofcyclohexane, filtered and dried to give 128 mg (38%) of compound 129.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 428.23; C₂₆H₄₂N₃O₂.

Example 14 3-Amino to 3-Acylamino Conversion in Steroid

Amide and sulfonamide analogues can be prepared from any amine relatedto compound 52. Scheme 13 shows the synthesis of the amide 131.Acetylation of the amine 52 in CH₂Cl₂, using acetyl chloride usingacetyl chloride and resin bound diethylamine gave the amide 130.Treatment with 80% acetic acid removed the acetonide group giving thedihydroxyamide 131.

Synthesis of Compound 130

A solution of the amine 52 (100 mg, 0.28 mmol), acetyl chloride (50 μl,0.70 mmol), 440 mg of PS-DIEA resin and 2.4 ml of CH₂Cl₂ was stirred atroom temperature for 16 hours. The resin was filtered and the filtratewas incubated for 2 hours with 260 mg of PS-Trisamine resin. The resinwas filtered and the filtrate was concentrated. Purification usingradial chromatography afforded 69 mg (62%) of compound 130.

Synthesis of Compound 131

A solution of the amide 130 (69 mg, 0.17 mmol) and 1 ml of 80% aceticacid was heated at 40° C. for 1 hour. The reaction mixture was twicetaken up and concentrated from 5 ml of toluene, once from 5 ml of MeOHand once from 1 ml of acetone and 5 ml of hexanes to give 62 mg (62%) ofcompound 131 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 384.16; C₂₂H₃₅NNaO₃, 362.20;C₂₂H₃₆NO₃, 344.18; C₂₂H₃₄NO₂.

Synthesis of Compound 132

Using the procedure described for the synthesis of compound 130, theamine 52 (88 mg, 0.24 mmol) was reacted with benzoyl chloride (65 μl,0.56 mmol) to give 64 mg of amide intermediate. A solution of the amideintermediate and 2 ml of 80% acetic acid was heated at 40° C. for 1hour. The reaction mixture was twice taken up and concentrated from 5 mlof toluene, once from 5 ml of MeOH and once from 1 ml of acetone and 5ml of hexanes to give 55 mg (55%) of compound 132 as a white solid (seeTable 3). LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1water and MeCN) 446.18; C₂₇H₃₇NNaO₃, 424.29; C₂₇H₃₈NO₃, 406.19;C₂₇H₃₆NO₂.

Synthesis of Compound 133

Using the procedure described for the synthesis of compound 130, theamine 52 (100 mg, 0.28 mmol) was reacted with isopropylsulfonyl chloride(6311, 0.56 mmol) to give 38 mg of sulfonamide intermediate. A solutionof the sulfonamide intermediate and 1.5 ml of 80% acetic acid was heatedat 40° C. for 1 hour. The reaction mixture was twice taken up andconcentrated from 5 ml of toluene, once from 5 ml of MeOH and once from1 ml of acetone and 5 ml of hexanes to give 35 mg (29%) of compound 133as an off-white solid (see Table 3). LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 426.14;C₂₃H₄₀NO₄S.

Synthesis of Compound 134

Using the procedure described for the synthesis of compound 130, theamine 52 (100 mg, 0.28 mmol) was reacted with benzenesulfonyl chloride(90 μl, 0.70 mmol) to give 105 mg of sulfonamide intermediate. Asolution of the sulfonamide intermediate and 2 ml of 80% acetic acid washeated at 40° C. for 5 hours. The reaction mixture was twice taken upand concentrated from 5 ml of toluene, once from 5 ml of MeOH and oncefrom 1 ml of acetone and 5 ml of hexanes to give 83 mg (65%) of compound134 as a white solid (see Table 3). LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 482.11; C₂₆H₃₇NNaO₄S, 477.17;C₂₆H₄₁N₂O₄S, 460.15; C₂₆H₃₈NO₄S.

Example 15

3-Acylamiobiotin-6,7-Hydroxy-17-Ethylidene Steroid

Scheme 14 shows the synthesis of the amide 135. Reaction of the amine 83with triethylamine and a water soluble version of biotin esterN-hydroxysuccinimide in methanol and water gave the biotinylated amideanalogue 135.

Synthesis of Compound 135

A solution of compound 83 (97 mg, 0.25 mmol), Et₃N (104 μl, 0.75 mmol),sulfo-NHS-biotin (120 mg, 0.27 mmol), 2.5 ml of MeOH and 2.5 ml of waterwas stirred at room temperature overnight. The reaction mixture wasconcentrated and purified using reverse phase column chromatographyeluting with 5% water/MeOH to afford 89 mg (64%) of compound 135 as anoff-white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAcin 4:1 water and MeCN) 560.30; C₃₁H₅₀N₃O₄S.

Example 16 3-Urea-6,7-Hydroxy-17-Methylidene Steroid

Any of the amines related to compound 52 can be reacted with isocyanatesor isothiocyanates to give compounds having urea or thioureafunctionalities. Compounds 136, 137 and 138 are examples of ureas thatwere synthesized using the methods shown in Scheme 15 (see Table 3).

Synthesis of Compound 136

A solution of the amine 52 (100 mg, 0.28 mmol), phenyl isocyanate (76μl, 0.70 mmol) and 2.4 ml of CH₂Cl₂ was stirred at room temperature for16 hours. The solution was incubated for 2 hours with 260 mg ofPS-Trisamine resin. The resin was filtered and the filtrate wasconcentrated. Purification using radial chromatography gave 95 mg (71%)of compound 136.

Synthesis of Compound 137

A solution of the urea 136 (95 mg, 0.20 mmol) and 2 ml of 80% aceticacid was heated at 80° C. for 2 hours. The reaction mixture was taken upand concentrated from 5 ml of toluene, from 5 ml of MeOH and from 5 mlof hexanes. Purification using radial chromatography eluting with 95:5:2CH₂Cl₂:MeOH:Et₃N afforded 40 mg (33%) of compound 137. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 461.18;C₂₇H₃₈N₂NaO₃, 439.22; C₂₇H₃₉N₂O₃, 421.25; C₂₇H₃₇N₂O₂.

Synthesis of Compound 138

Using the procedure described for the synthesis of compound 137, theamine 52 (100 mg, 0.28 mmol) was reacted with propyl isocyanate (52 μl,0.56 mmol) to give 72 mg of urea intermediate. A solution of the ureaintermediate and 2 ml of 80% acetic acid was heated at 80° C. for 2hours. The reaction mixture was twice taken up and concentrated from 5ml of toluene, once from 5 ml of MeOH and once from 5 ml of hexanes toafford 51 mg (45%) of compound 138 as a white solid. LC/MS (directinfusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 427.21;C₂₄H₄₀N₂NaO₃, 405.25; C₂₄H₄₁N₂O₃.

Example 17 3-Amino-6,7-Hydroxy-17-Dimethyl Unsaturated Steroid

Any compounds related to compounds 88 or 89 can undergo rearrangementusing the method shown in Scheme 16. Treatment of compound 88 with a 50°C. solution of hydrochloric acid in methanol and water removed theacetonide protecting group, facilitated migration of the 18-methyl groupto C17, and formed the ammonium chloride salt 139. Treatment of compound89 with the same conditions also gave compound 139. Example compounds140-148 were synthesized using the method shown in Scheme 16 (see Table4).

Synthesis of Compound 139

A solution of compound 88 (300 mg, 0.834 mmol), 4 drops of concentratedHCl, 2 ml of methanol and 2 ml of water was heated at 50° C. for 72hours. The reaction mixture was concentrated and the residue was twicetaken up in 5 ml of methanol and concentrated. The residue was taken upin 2 ml of methanol, diluted with 15 ml of acetone and concentrated. Theresidue was triturated in 5 ml of acetone, filtered and dried to give286 mg (96%) of compound 139 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 320.20; C₂₀H₃₄NO₂.

Synthesis of Compound 139

Using the same procedure as described for the synthesis of compound 139from compound 88, compound 89 was reacted to give 145 mg (77%) ofcompound 139 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 320.20; C₂₀H₃₄NO₂.

Synthesis of Compound 140

Using the procedure described for the synthesis of compound 99, theamine 88 (200 mg, 0.55 mmol) was reacted with m-tolualdehyde (90 μl,0.61 mmol). Purification using radial chromatography eluting with 5%MeOH/EtOAc gave 127 mg of amine intermediate. A solution of theintermediate amine, 4 drops of concentrated HCl, 1 ml of MeOH and 1 mlof water was heated at 50° C. for 20 hours. The reaction mixture wastaken up and concentrated thrice from 5 ml of MeOH and once from 5 ml ofacetone to give 74 mg (30%) of compound 140 as an off-white foam. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)424.24; C₂₈H₄₂NO₂.

Synthesis of Compound 141

Using the procedure described for the synthesis of compound 99, theamine 88 (200 mg, 0.55 mmol) was reacted with 3,4-difluorobenzaldehyde(67 μl, 0.61 mmol). Purification using radial chromatography elutingwith 30% EtOAc/hexanes gave 88 mg of amine intermediate. A solution ofthe intermediate amine, 4 drops of concentrated HCl, 1 ml of MeOH and 1ml of water was heated at 50° C. for 20 hours. The reaction mixture wastaken up and concentrated thrice from 5 ml of MeOH and once from 5 ml ofacetone to give 73 mg (28%) of compound 141 as an off-white foam. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)446.42; C₂₇H₃₈F₂NO₂.

Synthesis of Compound 142

Using the procedure described for the synthesis of compound 59, theamine 49 (200 mg, 0.55 mmol) was reacted with 3,4-dimethoxybenzaldehyde(70 μl, 0.61 mmol). Purification using radial chromatography elutingwith 40% EtOAc/hexanes gave 67 mg of amine intermediate. A solution ofthe intermediate amine, 4 drops of concentrated HCl, 1 ml of MeOH and 1ml of water was heated at 50° C. for 20 hours. The reaction mixture wastaken up and concentrated thrice from 5 ml of MeOH and once from 5 ml ofacetone to give 43 mg (16%) of compound 142 as yellow solid. LC/MS(direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN)470.27; C₂₉H₄₄NO₄.

Synthesis of Compound 143

A solution of compound 28 (200 mg, 0.540 mmol), 4 drops of concentratedHCl, and 3 ml of water was heated at 50° C. for 72 hours. The reactionmixture was concentrated and the residue was twice taken up in 5 ml ofmethanol and concentrated. The residue was taken up in 3 ml of methanol,diluted with 20 ml of acetone and concentrated. The residue wastriturated in 10 ml of acetone, filtered and dried to give 179 mg (90%)of compound 143 as a white solid. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 334.20; C₂₁H₃₆NO₂.

Synthesis of Compound 144

Titanium(IV) isopropoxide (270 μl, 0.92 mmol) was added to a roomtemperature solution of the ketone 108 (250 mg, 0.67 mmol), aniline (56μl, 0.61 mmol) and 1.5 ml of THF under argon. After 12 hours a solutionof NaBH₄ (65 mg, 1.7 mmol) in 2.3 ml of EtOH was added and the reactionwas continued for another 8 hours. The reaction was quenched by theaddition of 0.5 ml of water and filtered to remove a white precipitate.The solution was loaded onto a column of 600 mg of MP-TsOH resin andeluted with 9 ml of MeOH then 9 ml of 2 M NH₃ in MeOH. The NH₃/MeOHfraction was concentrated and the residue was taken up in 4 ml THF andtreated with 500 mg of PS-benzaldehyde resin and filtered to remove anyresidual aniline. The solution was concentrated and the residue wastaken up in 2 ml of 9:1 THF and water and 100 μl of concentrated HCl.After stirring at room temperature overnight the reaction mixture wasconcentrated. The residue was triturated in 1 ml of cyclohexane,filtered and dried to afford 62 mg of compound 144 as a white solid.LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAc in 4:1 water andMeCN) 410.03; C₂₇H₄₀NO₂.

Synthesis of Compound 145

Using the procedures described for the synthesis of compound 144,compound 108 (250 mg, 0.67 mmol) was reacted with3-(trifluoromethyl)aniline. The intermediate product was purified usingradial chromatography and then reacted with 100 μl of concentrated HClin 2 ml of 9:1 THF and water. After stirring at room temperatureovernight the reaction mixture was concentrated and the residue wastriturated in 1 ml of cyclohexane, filtered and dried to afford 23 mg ofcompound 145 as a white solid. LC/MS (direct infusion, electrospray +ve,10 mM NH₄OAc in 4:1 water and MeCN) 477.94; C₂₈H₃₉F₃NO₂.

Synthesis of Compound 146

Titanium(IV) isopropoxide (21611, 0.73 mmol) was added to a roomtemperature solution of the ketone 108 (200 mg, 0.54 mmol), benzylamine(53 μl, 0.49 mmol) and 1.2 ml of THF under argon. After 12 hours asolution of NaBH₄ (52 mg, 1.4 mmol) in 1.7 ml of EtOH was added and thereaction was continued for another 6 hours. The reaction was quenched bythe addition of 1 ml of water and filtered to remove a whiteprecipitate. The solution was diluted with 70 ml of CH₂Cl₂, washed with10 ml of water and 20 ml of brine, dried over MgSO₄, filtered andconcentrated. Purification using radial chromatography elutingconsecutively with 20% EtOAc/hexanes, EtOAc and 95:5:2 CH₂Cl₂/MeOH/Et₃Nafforded 127 mg of 3α-amine intermediate and 26 mg of 3β-amineintermediate. A solution of the 127 mg of 3α-amine intermediate, 1 ml of9:1 THF and water and 0.1 ml of concentrated HCl was stirred at roomtemperature overnight. The reaction mixture was concentrated, theresidue was taken up in 5 ml of MeOH and concentrated. The residue wastriturated in 1 ml of cyclohexane, filtered and dried to afford 118 mg(95%) of compound 146 as a white solid. LC/MS (direct infusion,electrospray +ve, 10 mM NH₄OAc in 4:1 water and MeCN) 424.20; C₂₈H₄₂NO₂.

Synthesis of Compound 147

A solution of the 26 mg of 3β-amine intermediate, the synthesis of whichwas described under the synthesis of compound 146, 1 ml of 9:1 THF andwater and 0.1 ml of concentrated HCl was stirred at room temperatureovernight. The reaction mixture was concentrated, the residue taken upin 5 ml of MeOH and concentrated. The residue was triturated in 1 ml ofcyclohexane, filtered and dried to afford 26 mg (100%) of compound 147as a white solid. LC/MS (direct infusion, electrospray +ve, 10 mM NH₄OAcin 4:1 water and MeCN) 424.21; C₂₈H₄₂NO₂.

Synthesis of Compound 148

A solution of compound 78 (63 mg, 0.18 mmol), 4 drops of concentratedHCl, 1 ml of methanol and 1 ml of water was heated at 50° C. for 48hours. The reaction mixture was concentrated and the residue was twicetaken up in 5 ml of methanol and concentrated. The residue was taken upin 2 ml of hexanes, concentrated and dried for 2 hours using anAbderhalden drying apparatus with refluxing acetone to give 69 mg (100%)of compound 148 as a white solid. LC/MS (direct infusion, electrospray+ve, 10 mM NH₄OAc in 4:1 water and MeCN) 348.20; C₂₂H₃₈NO₂. TABLE 1

3α- or Hex Calcium Com- 3β- IC₅₀ (% inhibition pound NH₂ R₁ R₂ R₃ HA(μM) at 20 μM) 49 α CH₃ H H HCl 11.0 26.8 53 α H H H AcOH 17.6 −0.8 54 αH H H HCl 16.1 19.8 64 α CH₃ F H HCl 14.4 6.7 69 α H CO₂CH₃ H AcOH 21.211.2 28 β CH₃ H H HCl 6.8 29.0 83 β CH₃ H H AcOH 7.7 30.3 89 β H H HAcOH 11.5 15.0 95 β CH₃ F H HCl 13.5 11.2 78 α H H CH₃ none 20.1 5.3 79α H H CH₃ HCl 18.4 9.1 80 α H H CH₃ AcOH 10.7 7.4

TABLE 2

Calcium Com- 3α- or 3β- Hex IC₅₀ (% inhibition pound NH₂ R₁ R₂ HA (μM)at 20 μM) 70 α O O AcOH ND 7.0 72 α OH H AcOH ND 10.3 96 β CH₃CH₂ H HCl7.8 47.9 97 α CH₃CH₂ H HCl 9.3 43.0

TABLE 3

Calcium 3α- or 3β- Hex IC₅₀ (% inhibition Compound NH₂ R₁ R₂ R₃ (μM) at20 μM) 131 α H H CH₃CO 14.1 9.9 132 α H H C₆H₅CO 10.0 19.6 133 α H H(CH₃)₂CHSO₂ 15.9 13.8 134 α H H C₆H₅SO₂ 16.7 25.1 135 β CH₃ H

15.1 37.3 137 α H H C₆H₅NHCO 14.8 13.0 138 α H H CH₃(CH₂)₂NHCO 15.0 9.0

TABLE 4

Calcium 3α- or 3β- Hex IC₅₀ (% inhibition Compound NH₂ R₁ R₂ R₃ R₄ (μM)at 20 μM) 139 β CH₃ CH₃ H H 10.3 26.3 140 α CH₃ CH₃ H 3-(CH₃)C₆H₄CH₂10.2 21.5 141 α CH₃ CH₃ H 3,4-(F)₂C₆H₃CH₂ 9.4 36.3 142 α CH₃ CH₃ H3,4-(CH₃O)₂C₆H₃CH₂ 15.9 13.8 143 β CH₃ CH₃CH₂ H H 8.6 22.6 144 α and βCH₃ CH₃CH₂ H C₆H₅ 6.9 13.0 145 α and β CH₃ CH₃CH₂ H 3-(CF₃)C₆H₄ 22.0−0.2 146 α CH₃ CH₃CH₂ H C₆H₅CH₂ 9.0 41.7 147 β CH₃ CH₃CH₂ H C₆H₅CH₂ 18.136.3 148 α CH₃ CH₃ CH₃ H 29.2 6.6

TABLE 5

Hex Calcium Com- IC₅₀ (% inhibition pound R HA (μM) at 20 μM) 110

AcOH 10.9 39.7 111

HCl 9.3 53.1 113 CH₃NH HCl 9.8 45.0 114 CH₃(CH₂)₂NH HCl 10.2 37.9 115CH₃(CH₂)₄NH HCl 11.1 40.0 116 C₅H₉NH AcOH 9.6 60.9 117 C₆H₁₁NH AcOH 6.642.2 118

AcOH 11.0 41.8 119 CH₃(CH₂)₂NH(CH₂)₂NH 2HCl 6.1 54.5 120 (CH₃)₂N(CH₂)₂NH2HCl 9.1 44.0 121

AcOH 14.4 42.4 122 HOCH₂CH₂NH HCl 12.3 31.4 123 HOCH₂(CH₂)₄NH HCl 16.712.8 124 HOCH₂CH₂NHCH₂CH₂NH 2HCl ND 21.0 125 3-(CH₃)C₆H₄NH₂ HCl 39.4 5.7126 4-(HO)C₆H₄NH HCl 9.7 45.0 127 4-(H₂NSO₂)C₆H₄NH HCl 10.1 29.4 128

AcOH 10.5 41.0 129

2AcOH N/A 13.8

TABLE 6

Calcium Hex IC₅₀ (% inhibition Compound R HA (μM) at 20 μM) 1004-((CH₃)₂CH)C₆H₄ AcOH 18.2 33.2 101 2-(F)C₆H₄ AcOH 10.3 16.3 1023-(CF₃)C₆H₄ AcOH 10.0 9.5 103 2-(CH₃O)C₆H₄ AcOH 11.1 23.7 1044-(CF₃O)C₆H₄ AcOH 9.5 30.3 105 3-(C₆H₅O)C₆H₄ AcOH 22.5 10.2 1063-(NO₂)C₆H₄ none 8.8 8.1 107 3-C₅H₄N 2HCl 17.2 11.6

UTILITY EXAMPLES Example A Effect of Selected Compounds onAllergen-Induced Lung Inflammation

The ability of a compound to inhibit the allergen-induced accumulationof inflammatory cells such as eosinophils and neutrophils in the lavagefluid obtained from sensitized animals is indicative of that compound'santi-asthma activity. In particular, this model system is useful in theevaluation of the effects of a test compound in the treatment of thelate phase response of asthma, when lung inflammation and the secondphase of bronchoconstriction is apparent. The test is conducted asfollows.

Male Brown Norway rats are sensitized to ovalbumin by singleintraperitoneal injection of 1 mg ovalbumin adsorbed to 100 mg Al(OH)₃(alum) in 1 ml sterile saline (saline control rats receive only sterilesaline) on day 1, and allowed to sensitize until day 21. Test compoundsare given orally q.d. for three days prior to challenge (days 19, 20,21), and one day post challenge (day 22), with the third dose given 2hours before challenge, and the fourth day dose given 24 hours afterchallenge (volume=300 μl/dose). Rats are challenged with 0.5% ovalbuminin saline generated using a Devillbis nebulizer for 60 min on day 21.

Forty-eight hrs after challenge, animals are sacrificed with an overdoseof intraperitoneally-delivered sodium pentobarbitol and the lungs arelavaged with cold 2×7 ml phosphate buffered saline. The recovered lavagefluid is placed on ice. The bronchoalveolar lavage fluid is centrifugedand the supernatant removed. The pellet is resuspended in phosphatebuffered saline at 4° C. Cytospins are prepared and stained fordifferentiation and enumeration of cell types.

The protective effects of the various test compounds on allergen inducedlung inflammation are summarized in Tables 7 and 8. The dose responseactivity of select compounds is shown in Table 9. Test compound wasadministered in 300 μl corn oil (Tables 7 and 8) or water (Table 9),which were used as vehicles. Control animals received 300 μl corn oil orwater alone, i.e., no drug. Values in Tables 7, 8, and 9 representpercent inhibition of leukocyte accumulation relative to controlanimals. A negative value in Table 7, 8, or 9 indicates an exacerbationof the effect over the control animal. TABLE 7 EFFECT OF TEST COMPOUNDS(5 MG/KG/DAY FOR 4 DAYS, P.O.) ON OVALBUMIN-INDUCED ACCUMULATION OFINFLAMMATORY CELLS IN THE LUNG LAVAGE FLUID OBTAINED FROM SENSITIZEDBROWN NORWAY RATS % inhibition of % inhibition of % inhibition ofCompound eosinophils neutrophils lymphocytes 83 67 34 38 97 40 52 56 96−9 40 19 64 62 70 81 89 57 60 64 28 85 87 124 53 24 57 13 95 14 45 30 4952 36 73 135 20 58 107

TABLE 8 EFFECT OF SELECTED COMPOUNDS (1 MG/KG/DAY FOR 4 DAYS, P.O.) ONOVALBUMIN-INDUCED ACCUMULATION OF INFLAMMATORY CELLS IN THE LUNG LAVAGEFLUID OBTAINED FROM SENSITIZED BROWN NORWAY RATS % inhibition of %inhibition of % inhibition of Analogue eosinophils neutrophilslymphocytes 142 −131 −1 −42 54 −82 −19 −7 107 −122 23 −28 124 −55 −76−26 129 −296 −114 −71 146 8 24 −27 147 40 58 36 131 16 57 21 138 −52 3533 133 −43 40 28

TABLE 9 DOSE DEPENDENT EFFECT OF SELECTED COMPOUNDS (MULTIPLE DOSING, 4DAYS QD, P.O.) ON OVALBUMIN-INDUCED ACCUMULATION OF INFLAMMATORY CELLSIN THE LUNG LAVAGE FLUID OBTAINED FROM SENSITIZED BROWN NORWAY RATS %inhibition of % inhibition of % inhibition of mg/kg eosinophilsneutrophils lymphocytes Analogue 1 0.3 0.1 0.03 1 0.3 0.1 0.03 1 0.3 0.10.03 89 71 80 35 — 66 72 25 — 6 70 2 — 28 60 71 29 5 41 66 −10 −26 70 8550 −5 139 42 41 46 — 63 62 42 — 56 34 19 — 143 48 29 28 — 64 25 57 — −22−37 −43 —

Example B Effect of Compound 83on Irritant-Induced Mouse Ear Edema

A number of mice are uniquely identified by placing a mark with anindelible marker on their tail. Mice are dosed orally with 15 mg/kg testcompound in 100 μl of 45% β-cyclodextrin in saline. Mice are brieflyanaesthesized with 2% halothane, and 2 μg of phorbol 12-myristate13-acetate in 25 μl of acetone is applied to the inner and outer sidesof the left ear of the mouse. Acetone is applied to the right ear of themouse in the same manner to serve as a vehicle control. Control animalsreceive the same treatment but without any test compound. After 3 hours,mice are sacrificed by cervical dislocation, and a standard sized biopsyis excised from the ears and weighed to the nearest 1/10th of a mg. Dataare analyzed by taking the difference of each left ear from the rightear, and then calculating the % inhibition of edema by (((mean Rx/meanirritant))×100)−100.

The compounds of the present invention demonstrate protective effects onirritant induced mouse ear edema. For example, compound 83 inhibitsirritant-induced mouse ear edema by 38% compared to control animals.

Example C Effect of Compounds on the Release of Hexosaminidase from aRat Mast Cell Line (RBL-2H3)

The anti-allergic effects of a compound of the present invention wasevaluated by measuring its effect on antigen-induced secretion ofhexosaminidase from a passively sensitized rat mast cell line. Theability of a test compound to inhibit the release of mast cell granulecontents, e.g., histamine and hexosaminidase, is indicative of theanti-allergy and/or anti-asthma activity of the compound. Hexosaminidaseis released from the mast cell granule along with histamine and othermediators during antigen challenge. The test is performed as follows.

RBL-2H3 cells are grown in culture and passively sensitized for 1 hourat 37° C. to dinitrophenol (DNP) using anti-human-DNP (IgE). Cells areincubated with test compound for 30 minutes at 37° C. and stimulatedwith 0.5 μg/ml DNP-HSA (antigen) for 30 minutes. Aliquots of thesupernatant are removed and used to measure the amount of hexosaminidasereleased during challenge with the antigen. The amount of hexosaminidasepresent in the supernatant is determined calorimetrically by monitoringthe enzymatic metabolism of p-nitrophenyl-N-acetyl-β-D-glucosaminide(p-NAG) over a period of 60 minutes at 405 nm. The effect of each testcompound is determined as a percentage of the antigen-induced response(minus background release) obtained in the presence of DMSO alone. Thesevalues are used to determine the degrees of inhibition ofantigen-induced hexosaminidase release from the cells.

The compounds of the present invention demonstrate the ability toinhibit hexosaminidase release in response to antigen stimulation.Compounds were tested at 0.3, 3, 10 and 30 μM, and the IC₅₀ calculated.This data is summarized in Tables 1-6. For example, compound 119 at 6.1μM inhibits hexosaminidase release by 50% in response to antigenstimulus.

Example D Metabolic Stability of Selected Compounds in Human S9Fractions

The therapeutic effectiveness of a test compound can be often directlyrelated to its metabolic stability in vivo. The majority of known,marketed drugs are metabolized by a group of enzymes known as P450enzymes. The S9 fraction of human liver contains all the P450 enzymesand also cytoplasmic enzymes that may be involved in the metabolism ofnew chemical entities. Metabolism in vitro using human S9 fractions is astandard assay to evaluate relative metabolic stability of newcompounds. The test is performed as follows.

Reagents are thawed on ice and combined to make up a Master Mix asfollows: Potassium phosphate (100 mM, pH 7), G6P (0.25 mM), G6PDH (2U/ml), NADPH (1 mM), UDP (0.25 mM), APPS (0.25 mM), and S9 fraction (2mg/ml). A volume of 498 μl of the Master mix is dispensed into eachmicrocentrifuge tube. A volume of 2 μl of 2.5 mM test compound (finalconcentration of 10 μM) is dispensed into the center of the lid of theappropriate tube, and the lids gently closed to prevent mixing.Synchronous combination of test compound and master mix is achieved bysimultaneous ten times inversion of the tubes, which are then incubatedat 37° C. and shaken at 150 rpm for the appropriate incubation times.While incubation is in progress, all 0 time samples are mixedindividually by three times inversion followed by the immediate additionof 500 μl ice cold acetonitrile with three times inversion to stop thereaction. Immediately after each 15 minutes and 30 minutes incubation iscomplete, ice cold acetonitrile is added to each tube and the tubesmixed by three times inversion. All sample tubes are incubated at −80°C. for a minimum of 15 minutes, thawed and remixed by inversion.Aliquots of 650 μl are transferred to Chromatographic Specialtiesmicro-spin filter system tubes (0.2 μm nylon membrane, C618505) andcentrifuged at 13,000 rpm for 48 seconds. The sample filtrates arestored at −80° C.

Sample filtrates are analyzed on LCMS, and percent remaining after 15and 30 minute incubation is calculated relative to the 0 minuteincubation.

The metabolic stability of various compounds are summarized in Table 10as the percent remaining after 15 or 30 minutes incubation with humanliver S9 fractions. TABLE 10 METABOLIC STABILITY OF SELECTED COMPOUNDSAFTER 15 AND 30 MINUTES INCUBATION WITH HUMAN LIVER S9 FRACTIONS SHOWNAS THE PERCENT REMAINING OF STARTING CONCENTRATION AFTER 0 MINUTESINCUBATION Compound % remaining after 15 min % remaining after 30 min 8392 ± 19 91 ± 21 97 94 ± 10 89 ± 12 96 92 ± 19 94 ± 21 89 100 ± 17  98 ±16 28 100 ± 6  104 ± 12  49 93 ± 6  85 ± 8  64 104 ± 4  88 ± 5  139 109± 26  100 ± 10  143 100 ± 3  99 ± 10 146 82 ± 6  63 ± 9  107 84 ± 5  62± 6  142 80 ± 11 55 ± 2  69 73 ± 24 63 ± 6  104 53 ± 10 56 ± 18 141 100± 9  26 ± 4  134 85 ± 54 36 ± 15 137 93 ± 31 71 ± 17 138 71 ± 4  65 ± 65132 60 ± 17 40 ± 9  79 67 ± 6  61 ± 14

Example E Solubility of Selected Compounds in Physiologically CompatibleFormulations

Compounds of the present invention exhibit good water solubility. Forexample, compound 83 is soluble in water at 225 mg/ml. Compound 83substituted with a hydroxyl at C3 has a solubility in water of less than60 μg/ml. Compounds 28 and 89 have solubilities at room temperature of30 mg/ml, which can be significantly increased by heating. Thisunexpected finding indicates that these 3-amino compounds should bereadily formulated into therapeutic compositions.

Example F Effect of Selected Test Compounds on Antigen-Induced CalciumFlux

Elevation of cytoplasmic calcium concentration is a common and crucialevent which follows the activation of many types of cell surfacereceptors. Increases in intracellular calcium that occur followingagonist activation of inositol lipid hydrolysis are the results ofcalcium release from the endoplasmic reticulum and the influx of calciumthrough the plasma membrane. Increases in cytosolic calciumconcentration are involved in many important cellular responses in theinflammatory process including adhesion, motility, gene expression,proliferation, and degranulation. Changes in intracellular calciumconcentrations can affect both short and long term cellular responses.An assay method to evaluate a test compound's impact on calcium flux isprovided as follows.

Jurkat clone E6.1 cells grown in RPMI medium supplemented with 10% FBSand 2 mM L-Glutamine are transferred to 50 mL conical tubes andcentrifuged for 5 minutes at 900 RPM to form a cell pellet. Theresulting supernatants are discarded and each pellet is washed in 10 mLHBSS. Cell suspensions are accumulated and centrifuged for 5 minutes at900 RPM to form a cell pellet. The resulting supernatant is discardedand the pellet is resuspended in HBSS at 1×10⁷ cells/mL. The cellsuspension is transferred to a 20 mm petri dish and incubated at 37° C.,5% CO₂ for 20 minutes.

One volume of Fura 2AM is mixed to one volume of detergent PluronicF127. Cells are labeled with 4 μL of probe solution to each mL of cellsuspension. The petri dish is wrapped in aluminum foil to protect fromlight and placed on a plate shaker for 30 minutes at room temperature.

The following steps are done in the laminar flow hood with thefluorescent lights turned off. The petri dish is removed from the shakerand the labeled cell suspension is transferred to a 15 mL conical tubeand washed twice with HBSS as above. The cell pellet is resuspended in12 mL HBSS and left wrapped in foil for 30 minutes at room temperature.The labeled cell suspension is aliquoted (100 μL/well) into a Dynex 96well white opaque tissue culture plate. 50 μL of each test sample isadded to appropriate wells and are incubated together for 10 minutes at37° C. in the Wallac 1920 Victor™ plate reader (test samples areprepared in HBSS at 20 mM, final concentration is 20 μM). Selectedcompounds were tested for dose related activity. Test samples andactivator (anti-CD3 mAb at final concentration of 4 μg/mL, PharMingen)are added manually (50 μL), such that the minimum time to acquire thefirst data point after stimulation is approximately 30 seconds. Thecalcium influx response to anti-CD3 mAb is measured as an “end point”assay. The entire plate is read in 100 seconds using a kinetic of 1second per well.

The plate is read before the addition of anti-CD3 to monitornon-specific effect of samples/drugs. Fluorescence emission is measuredat 510 nm with excitation alternating between 340 and 380 every secondusing an excitation/emission filter pair. Data from these dualwavelengths are represented as a ratio of 2 excitations wavelengths.This ratio is independent of intracellular dye and cell concentrations,enabling real comparison between experiments.

The effect of selected compounds on calcium flux in antigen challengedJurkat clone E6.1 cells is summarized in Tables 1-6. For example,compound 116 inhibited calcium by 60.9% at 20 μM. The IC₅₀ for compound119 is less than or equal to 10 μM when dose response activity wasexamined. This demonstrated substantial effect on calcium flux would bebeneficial in any disease pathology for which calcium is a significantsecond messenger or effector molecule, including but not limited toischemia/reperfusion injury such as stroke or myocardial infarction,inflammatory diseases such as asthma or allergy, neural or musculardisorders such as Parkinson's disease or epilepsy, cardiac arrhythmias,or hypertension.

Example G Effect of Selected Compounds on Allergen-Induced Changes inLung Function

In asthma, the early response of the airways to allergen challenge ischaracterized by an immediate bronchoconstriction which peaks 20-30 minafter exposure to the stimuli, and which normally resolves afterapproximately 2 hours. Anti-inflammatories are not generally activebronchodilators and are not very effective in the control of acuteasthmatic bronchoconstriction. This results in the need for combinationtherapy to treat both the bronchoconstriction and the inflammation.

Cam-Hartley guinea pigs were sensitized to ovalbumin (OA) in groups of5-6 by exposure to an aerosolized solution of 1% OA in saline for 15 minon 2 consecutive days via a DeVilbiss nebulizer, with an additionalsingle intradermal injection of 3 μg OA in saline on Day 1. Animals werefound to be at peak sensitivity to the antigen approximately 14 daysafter the initial exposure. On Day 14, the animals were initiallyanaesthetized with ketamine (50 mg/kg i.p.) and xylazine (10 mg/kgi.p.), weighed, and then maintained on 1% halothane delivered via a nosecone. The left carotid artery was cannulated with PE90 tubing containing200 U/ml heparin in saline. A tracheostomy was performed and afluid-filled cannula (PE 160) was inserted approximately 7 cm into theesophagus. The animal was positioned in a plethysmograph and the tracheaattached to a fixed stainless steel tracheal tube in the body box. Thecarotid cannula was attached to a pressure transducer for monitoring ofblood pressure and heart rate. The guinea pig was paralyzed withpancuronium bromide (0.8 mg/kg) and ventilated with air at a frequencyof 60 Hz and tidal volume of 3 ml using a Harvard small animalventilator.

Data was collected for 20 sec periods at a sampling rate of 100 Hz on acomputer-linked physiological recording system using DIREC physiologicalsoftware and analyzed using ANADAT software. Pulmonary resistance anddynamic lung compliance values were obtained from the volume, flow andpressure signals according to the method of Von Neeguard & Wirz (1927),using an isovolumetric multi-point regression model for analysis(Ludwig, Robatto, et al. 1991), and calculated as absolute changes inlung resistance (R_(L); cm H₂O/ml/s) or lung compliance (C_(DYN);ml/cmH₂O). Volume and pressure signals were calibrated before each setof experiments following standard procedures.

Several lung function measurements were obtained over a 5-10 minuteperiod to ensure a steady baseline, and then the animal was challengedwith OA (2% in saline) administered in 6 tidal breaths as a nebulizedaerosol at a flow rate of 5 L/min. Pulmonary and cardiovascular functionwas continually monitored throughout the experiment, although data wascollected at specific time-points after antigen challenge (10 s, 1, 2,3, 4, 5, 10, 20, and 30 min).

Test compounds were administered under light halothane anesthesia byoral gavage (0.1-1.0 mg/kg/day q.d.) in 300 μl polyethylene glycol-200for 4 days prior to challenge with the final dose administered 2 hoursprior to antigen challenge.

The protective effects of select test compounds on allergen inducedbronchoconstriction are summarized in FIGS. 4 and 5. The duration ofactivity of Compound 89 is shown in FIGS. 6 and 7. Data is presented asmean±standard error of the mean. The inhibition of thebronchoconstriction by the test compounds would be beneficial in anydisease where acute smooth muscle constriction in response to allergenchallenge is manifest, such as asthma and allergy.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference. For example, the book inComprehensive Organic Transformations, A Guide to Functional GroupPreparations, Second Edition, Richard C. Larock, John Wiley and Sons,Inc., 1999, and particularly the references cited therein, isincorporated herein by reference for all purposes.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound of the formula

and pharmaceutically acceptable salts, solvates, stereoisomers andprodrugs thereof, in isolation or in mixture, where independently ateach occurrence: R¹ and R² are selected from hydrogen, oxygen so as toform nitro or oxime, amino, —SO₃—R, and organic groups having 1-30carbons and optionally containing 1-6 heteroatoms selected fromnitrogen, oxygen, phosphorous, silicon, and sulfur, where R² may be adirect bond to numeral 3, or R¹ and R² may, together with the N to whichthey are both bonded, form a heterocyclic structure that may be part ofan organic group having 1-30 carbons and optionally containing 1-6heteroatoms selected from nitrogen, oxygen and silicon; or R¹ may be a 2or 3 atom chain to numeral 2 so that —N—R¹—forms part of a fusedbicyclic structure to ring A; R³ and R⁴ are selected from direct bondsto 6 and 7 respectively so as to form carbonyl groups, hydrogen, or aprotecting group such that R³ and/or R⁴ is part of hydroxyl or carbonylprotecting group; numerals 1 through 17 each represent a carbon, wherecarbons at numerals 1, 2, 4, 11, 12, 15, 16 and 17 may be independentlysubstituted with (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or (b) two of the following, which are independentlyselected: —X, —N(R¹)(R²), —R⁵ and —OR⁶; and where carbons at numerals 5,8, 9, 10, 13 and 14 may be independently substituted with one of —X,—R⁵, —N(R¹)(R²) or —OR⁶; in addition to the —OR³ and —OR⁴ groups asshown, each of carbons 6 and 7 may be independently substituted with oneof —X, —N(R¹)(R ), —R⁵ or —OR⁶; each of rings A, B, C and D isindependently fully saturated, partially saturated or fully unsaturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded; R⁶ is H or aprotecting group such that —OR⁶ is a protected hydroxyl group, wherevicinal —OR⁶ groups may together form a cyclic structure that protectsvicinal hydroxyl groups, and where geminal —OR⁶ groups may together forma cyclic structure that protects a carbonyl group; and X representsfluoride, chloride, bromide and iodide.
 2. A compound of claim 1 whereinnumerals 1 through 16 each represent a carbon, where carbons at numerals1, 2, 4, 11, 12, 15 and 16 may be independently substituted with (a) oneof: ═O, ═C(R ⁵)(R ⁵), ═C═C(R⁵)(R⁵), —C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)—and—(O(C(R⁵)(R⁵))_(n)O)— wherein n ranges from 1 to about 6; or (b) two ofthe following, which are independently selected: —X, —N(R¹)(R²), —R⁵ and—OR⁶; and numeral 17 represents a carbon substituted with (a) one of:═C(R^(5a))(R^(5a)), ═C═C(R^(5a))(R^(5a)), and—C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)— wherein n ranges from 1 toabout 6; or (b) two of the following, which are independently selected:—X, —N(R¹)(R²), and —R^(5a); where R^(5a) at each occurrence isindependently selected from H, X, and C₁₋₃₀ organic moiety that mayoptionally contain at least one heteroatom selected from the groupconsisting of boron, halogen, nitrogen, silicon and sulfur; where twogeminal R⁵ groups may together form a ring with the carbon atom to whichthey are both bonded.
 3. A compound of claim 2 wherein R^(5a) at eachoccurrence is independently selected from C₁₋₃₀ hydrocarbon, C₁₋₃₀halocarbon, C₁₋₃₀ hydrohalocarbon, H, and X.
 4. A compound of claim 2wherein R^(5a) at each occurrence is independently selected from C₁₋₁₀hydrocarbon, C₁₋₁₀ halocarbon, C₁₋₁₀ hydrohalocarbon, H, and X.
 5. Acompound of any of claims 1-4 wherein R¹ and R² are selected fromhydrogen, oxygen so as to form nitro or oxime, amino, —SO₃—R, andorganic groups having 1-30 carbons and optionally containing 1-6heteroatoms selected from oxygen, phosphorous, silicon, and sulfur,where R² may be a direct bond to numeral 3, or R¹ and R² may, togetherwith the N to which they are both bonded, form a heterocyclic structurethat may be part of an organic group having 1-30 carbons and optionallycontaining 1-6 heteroatoms selected from oxygen and silicon; or R¹ maybe a 2 or 3 atom chain to numeral 2 so that —N—R¹—forms part of a fusedbicyclic structure to ring A.
 6. A compound of any of claims 1-5 whereincarbons at numerals 1, 2, 4, 11, 12, 15 and 16 are each substituted withtwo hydrogens unless said carbon is part of an unsaturated bond; carbonsat numerals 5, 8, 9 and 14 are each substituted with one hydrogen unlesssaid carbon is part of an unsaturated bond; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond.
 7. A compound of any ofclaims 1-6 wherein carbons at numerals 1, 2, 4, 11, 12, 15 and 16 areeach substituted with two hydrogens; carbons at numerals 5, 8, 9 and 14are each substituted with one hydrogen; carbon at numeral 10 issubstituted with methyl; and carbon at number 13 is substituted withmethyl unless it is part of an unsaturated bond.
 8. A compound of claim1 wherein R¹ and R² are hydrogen; R³ and R⁴ are selected from directbonds to 6 and 7 respectively so as to form carbonyl groups, hydrogen,or a protecting group such that R³ and/or R⁴ is part of hydroxyl orcarbonyl protecting group; and in addition to the —OR³ and —OR⁴ groupsas shown, each of carbons 6 and 7 is substituted with hydrogen unlessprecluded because —OR³ or —OR⁴ represent a carbonyl group; carbons atnumerals 1, 2, 4, 11, 12, 15 and 16 are each substituted with twohydrogens unless said carbon is part of an unsaturated bond; carbons atnumerals 5, 8, 9 and 14 are each substituted with one hydrogen unlesssaid carbon is part of an unsaturated bond; carbon at numeral 10 issubstituted with methyl; carbon at number 13 is substituted with methylunless it is part of an unsaturated bond; carbon at numeral 17 issubstituted with (a) one of: ═O, ═C(R⁵)(R⁵), ═C═C(R⁵)(R⁵),—C(R⁵)(R⁵)(C(R⁵)(R⁵))_(n)— and —(O(C(R⁵)(R⁵))_(n)O)— wherein n rangesfrom 1 to about 6; or (b) two of the following, which are independentlyselected: —X, —N(R¹)(R²), —R⁵ and —OR⁶; each of rings A, B, C and D isindependently fully saturated, partially saturated or fully unsaturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur; where two geminal R⁵ groups may together form a ringwith the carbon atom to which they are both bonded; R⁶ is H or aprotecting group such that —OR⁶ is a protected hydroxyl group, wherevicinal —OR⁶ groups may together form a cyclic structure that protectsvicinal hydroxyl groups, and where geminal —OR⁶ groups may together forma cyclic structure that protects a carbonyl group; and X representsfluoride, chloride, bromide and iodide.
 9. A compound of claim 8 whereinR¹ and R² are hydrogen; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of the following,which are independently selected: —X, —N(R¹)(R²), and —R⁵; each of ringsA, B, C and D is independently fully saturated or partially saturated;R⁵ at each occurrence is independently selected from H, X, and C₁₋₃₀hydrocarbons, halocarbons and halohydrocarbons; and X representsfluoride, chloride, bromide and iodide.
 10. A compound of claim 9wherein R¹ and R² are hydrogen; R³ and R⁴ are selected from hydrogen andprotecting groups such that R³ and/or R⁴ is part of hydroxyl protectinggroup; carbons at numerals 1, 2, 4, 11, 12, 15 and 16 are eachsubstituted with two hydrogens; carbons at numerals 5, 8, 9 and 14 areeach substituted with one hydrogen; carbon at numeral 10 is substitutedwith methyl; carbon at number 13 is substituted with methyl unless it ispart of an unsaturated bond; carbon at numeral 17 is substituted with(a) one of: ═C(R⁵)(R⁵); or (b) two of —R⁵; each of rings A, B, C and Dis independently fully saturated or partially saturated; and R⁵ at eachoccurrence is independently selected from H and C₁₋₁₀ hydrocarbons. 11.A compound of any one of claims 1-10 of the formula


12. A compound of any one of claims 1-10 of the formula


13. A compound of any one of claims 1-10 of the formula


14. A compound of any one of claims 1-10 of the formula


15. A compound of any one of claims 1-10 of the formula


16. A compound of any one of claims 1-10 of the formula


17. A compound of claim 1 wherein 17 is substituted with ═C(R⁵)(R⁵) andR⁵ is selected from hydrogen, halogen, C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, and—CO₂—C₁₋₆alkyl.
 18. A compound of claim 1 wherein 17 is substituted withC₁₋₆alkyl or C₁₋₆haloalkyl.
 19. A compound of claim 1 wherein 17 issubstituted with —OR⁶ or ═O, wherein R⁶ is hydrogen.
 20. A compound ofclaim 1 wherein R¹ is selected from —C(═O)—R⁷, —C(═O)NH—R⁷; —SO₂—R⁷;wherein R⁷ is selected from alkyl, heteroalkyl, aryl and heteroaryl. 21.A compound of claim 20 wherein R⁷ is selected from C₁₋₁₀hydrocarbyl. 22.A compound of claim 20 wherein R⁷ comprises biotin.
 23. A compound ofclaim 1 wherein (R¹)(R²)N— is selected from


24. A compound of claim 1 wherein R¹ is hydrogen and R² comprises acarbocycle.
 25. A compound of claim 24 wherein the carbocycle is phenyl.26. A compound of claim 25 wherein R² is selected from 3-methylphenyl;4-hydroxyphenyl; and 4-sulfonamidephenyl.
 27. A compound of claim 1wherein R¹ is hydrogen and R² comprises a C₁₋₁₀hydrocarbyl.
 28. Acompound of claim 1 wherein R¹ is hydrogen and R² is heteroalkyl.
 29. Acompound of claim 28 wherein R² is selected fromC₁₋₁₀alkyl-W—C₁₋₁₀alkylene- wherein W is selected from O and NH;HO—C₁₋₁₀alkylene-; and HO—C₁₋₁₀alkylene-W—C₁₋₁₀alkylene- where W isselected from O and NH.
 30. A compound of claim 1 wherein R¹ is hydrogenand R² is —CH₂—R⁷ wherein R⁷ is selected from alkyl, heteroalkyl, aryland heteroaryl.
 31. A compound of claim 30 wherein R⁷ is selected fromalkyl-substituted phenyl; halogen-substituted phenyl; alkoxy-substitutedphenyl; aryloxy-substituted phenyl; and nitro-substituted phenyl.
 32. Acompound of claim 1 wherein each of R¹ and R² is hydrogen.
 33. Acompound of claims 1 or 32 wherein each of R³ and R⁴ is hydrogen.
 34. Acompound of claims 32 or 33 where the carbon at numeral 17 issubstituted with (a) one of the following: C(R^(5a))(R^(5a)),═C═C(R^(5a))(R^(5a)), and —C(R^(5a))(R^(5a))(C(R^(5a))(R^(5a)))_(n)—wherein n ranges from 1 to about 6; or (b) two of the following, whichare independently selected: —X, —N(R¹)(R²), and —R^(5a); where R^(5a) ateach occurrence is independently selected from H, X, and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, silicon and sulfur;where two geminal R⁵ groups may together form a ring with the carbonatom to which they are both bonded.
 35. A compound of claim 1 wherein R³and R⁴ together form a ketal of the structure


36. A compound of claim 1 wherein —OR³ and —OR⁴ have the stereochemistryshown


37. A compound of claim 1 wherein —N(R¹)(R²) is in a salt form.
 38. Acompound of claim 1 wherein —N(R¹)(R²) is in a salt form and the salt isa halogen or acetate salt.
 39. A compound of claim 1 which is a prodrugof the formula shown in claim
 1. 40. A compound of claim 1 andpharmaceutically acceptable salts, solvates, stereoisomers but notprodrugs thereof, in isolation or in mixture.
 41. A compound of claim 1wherein at least one of the carbons at numerals 10 and 13 aresubstituted with methyl.
 42. A compound of claim 1 wherein each of R¹and R² are independently selected from hydrogen and organic groupshaving 1-20 carbons and optionally containing 1-5 heteroatoms selectedfrom nitrogen, oxygen, silicon, and sulfur.
 43. A compound of claim 1wherein R¹ and R² are independently selected from hydrogen, R⁸, R⁹, R¹⁰,R¹¹ and R¹² where R⁸ is selected from alkyl, heteroalkyl, aryl andheteroaryl; R⁹ is selected from (R⁸)_(r)-alkylene,(R⁸)_(r)-heteroalkylene, (R⁸)_(r)-arylene and (R⁸)_(r)-heteroarylene;R¹⁰ is selected from (R⁹)_(r)-alkylene, (R⁹)_(r)-heteroalkylene,(R⁹)_(r)-arylene, and (R⁹)_(r)-heteroarylene; R¹¹ is selected from(R¹⁰)_(r)-alkylene, (R¹⁰)_(r)-heteroalkylene, (R¹⁰)_(r)-arylene, and(R¹⁰)_(r)-heteroarylene, R¹² is selected from (R¹¹)_(r)-alkylene,(R¹¹)_(r)-heteroalkylene, (R¹¹)_(r)-arylene, and(R¹¹)_(r)-heteroarylene, and r is selected from 0, 1, 2, 3, 4 and 5,with the proviso that R¹ and R² may join to a common atom so as to forma ring with the common atom.
 44. A compound of claims 1 or 43 wherein R³and R⁴ are selected from hydrogen and protecting groups such that R³and/or R⁴ is part of hydroxyl protecting group; carbons at numerals 1,2, 4, 11, 12, 15 and 16 are each substituted with two hydrogens unlesssaid carbon is part of an unsaturated bond; carbons at numerals 5, 8, 9and 14 are each substituted with one hydrogen unless said carbon is partof an unsaturated bond; carbon at numeral 10 is substituted with methyl;carbon at number 13 is substituted with methyl unless it is part of anunsaturated bond; carbon at numeral 17 is substituted with (a) one of:═C(R⁵)(R⁵) and ═C═C(R⁵)(R⁵); or (b) two of —R⁵; each of rings A, B, Cand D is independently fully saturated or partially saturated; and R⁵ ateach occurrence is independently selected from H and C₁₋₁₀ hydrocarbons.45. A compound of claims 1, 43 or 44 wherein R¹ and R² are independentlyselected from hydrogen, R⁸, R⁹, R¹⁰, R¹¹ and R¹² where R⁸ is selectedfrom C₁₋₁₀alkyl,C₁₋₁₀heteroalkyl comprising 1, 2 or 3 heteroatoms,C₆₋₁₀aryl and C₃₋₁₅heteroaryl comprising 1, 2 or 3 heteroatoms; R⁹ isselected from (R⁸)_(r)—C₁₋₁₀alkylene, (R⁸)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁸)_(r)—C₆₋₁₀arylene and(R⁸)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R¹⁰ isselected from (R⁹)_(r)—C₁₋₁₀alkylene, (R⁹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R⁹)_(r)—C₆₋₁₀arylene, and(R⁹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms; R₁₁ isselected from (R¹⁰)_(r)—C₁₋₁₀alkylene, (R¹⁰)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹⁰)—C₆₋₁₀arylene, and(R¹⁰)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, R¹² isselected from (R¹¹)_(r)—C₁₋₁₀alkylene, (R¹¹)_(r)—C₁₋₁₀heteroalkylenecomprising 1, 2 or 3 heteroatoms, (R¹¹)_(r)C₆₋₁₀arylene, and(R¹¹)_(r)—C₃₋₁₅heteroarylene comprising 1, 2 or 3 heteroatoms, and r isselected from 0, 1, 2, 3, 4 and 5, with the proviso that R¹ and R² mayjoin to a common atom so as to form a ring with the common atom.
 46. Acompound of claims 1, 43 or 44 wherein R¹ and R² are selected fromhydrogen, CH₃—, CH₃(CH₂)₂—, CH₃(CH₂)₄—, CH₃CO—, C₆H₅CO—(CH₃)₂CHSO₂—,C₆H₅SO₂—, C₆H₅NHCO—, CH₃(CH₂)₂NHCO—, CH₃(CH₂)₂NH(CH₂)₂—, (CH₃)₂N(CH₂)₂—,HOCH₂CH₂—, HOCH₂(CH₂)₄—, HOCH₂CH₂NHCH₂CH₂—, 3-(CH₃)C₆H₄—, 4-(HO)C₆H₄—,4-(H₂NSO₂)C₆H₄—, 4-((CH₃)₂CH)C₆H₄—CH₂—, 2-(F)C₆H₄—CH₂—,3-(CF₃)C₆H₄—CH₂—, 2-(CH₃O)C₆H₄—CH₂—, 4-(CF₃O)C₆H₄—CH₂—,3-(C₆H₅O)C₆H₄—CH₂—, 3-(NO₂)C₆H₄—CH₂—,

or R¹ and R² may join together with the nitrogen to which they are bothattached and form a heterocycle selected from:


47. A compound of claims 1 or 43 of the formula


48. A compound of claims 1 or 43 of the formula


49. A compound of claims 1 or 43 of the formula


50. A compound of claims 1 or 43 of the formula


51. A compound of claim 1 wherein R¹ is a 2, or 3 atom chain to numeral2 so that —N—R¹— forms part of a fused bicyclic structure to ring A, thecompound having the formula:

where Z represents 2 or 3 atoms, independently selected from C, N and Oso long as a stable structure results, and the ring including Z may besaturated or unsaturated.
 52. A compound of claim 51 selected from


53. A pharmaceutical composition comprising a compound of any of claims1-52 and a pharmaceutically acceptable carrier, excipient or diluent.54. A method of treating inflammation therapeutically comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 55. A method of treatinginflammation prophylactically comprising administering to a subject inneed thereof a prophylactically-effective amount of a compound of any ofclaims 1-52.
 56. A method of treating asthma comprising administering toa subject in need thereof a therapeutically-effective amount of acompound of any of claims 1-52.
 57. A method of treating allergicdisease including but not limited to dermal and ocular indicationscomprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.58. A method of treating chronic obstructive pulmonary diseasecomprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.59. A method of treating atopic dermatitis comprising administering to asubject in need thereof a therapeutically-effective amount of a compoundof any of claims 1-52.
 60. A method of treating solid tumours comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 61. A method of treatingAIDS comprising administering to a subject in need thereof atherapeutically-effective amount of a compound of any of claims 1-52.62. A method of treating ischemia reperfusion injury comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of a compound of any of claims 1-52.
 63. A method of treatingcardiac arrhythmias comprising administering to a subject in needthereof a therapeutically-effective amount of a compound of any ofclaims 1-52.